Food Anal.
Methods (2015) 8:255271
DOI 10.1007/s12161-014-9915-6
Rapid Methods for Quality Assurance of Foods: the Next Decade
with Polymerase Chain Reaction (PCR)-Based Food Monitoring
D. De Medici & T. Kuchta & R. Knutsson & A. Angelov & B. Auricchio & M. Barbanera &
C. Diaz-Amigo & A. Fiore & E. Kudirkiene & A. Hohl & D. Horvatek Tomic & V. Gotcheva &
B. Popping & E. Prukner-Radovcic & S. Scaramaglia & P. Siekel & K. A. To & M. Wagner
Received: 25 February 2014 / Accepted: 10 June 2014 / Published online: 2 July 2014
# Springer Science+Business Media New York 2014
Abstract Microbiological analysis is an integral part of food
quality control, as well as of the management of food chain
safety. Microbiological testing of foodstuffs complements the
preventive approach to food safety activities based mainly on
implementation and application of the concept of Hazard
Analysis and Critical Control Points (HACCP). Traditional
microbiological methods are powerful but lengthy and cum-
bersome and therefore not fully compatible with current re-
quirements. Even more, pathogens exist that are fastidious to
cultivate or uncultivable at all. Besides immunological tests,
molecular methods, specifically those based on polymerase
chain reaction (PCR), are available options to meet industry
and enforcement needs. The clear advantage of PCR over all
other rapid methods is the striking analytical principle that is
based on amplification of DNA, a molecule being present in
every cell prone to multiply. Just by changing primers and
D. De Medici (*) : B. Auricchio : A. Fiore
Department of Veterinary Public Health and Food Safety, Istituto
Superiore di Sanit, Viale Regina Elena 299, 00161 Rome, Italy
e-mail: dario.demedici@iss.it
T. Kuchta : P. Siekel
Department of Microbiology and Molecular Biology,
Food Research Institute, Priemyseln 4, P. O. Box 25,
Bratislava 82475 26, Slovakia
R. Knutsson
Security Department, SVA National Veterinary Institute, 751
89 Uppsala, Sweden
A. Angelov : V. Gotcheva
Department of Biotechnology, University of Food Technologies, 26
Maritza Blvd, 4002 Plovdiv, Bulgaria
M. Barbanera : S. Scaramaglia
Laboratorio Coop Italia, 40033 Casalecchio di Reno, Bologna, Italy
C. Diaz-Amigo : B. Popping
Eurofins CTC GmbH, Stenzelring 14b, 21107 Hamburg, Germany
probes, different genomes such as bacteria, viruses or parasites
can be detected. A second advantage is the ability to both
detect and quantify a biotic contaminant. Some previously
identified obstacles of implementation of molecular methods
have already been overcome. Technical measures became
available that improved robustness of molecular methods,
and equipment and biochemicals became much more afford-
able. Unfortunately, molecular methods suffer from certain
drawbacks that hamper their full integration to food safety
control. Those encompass a suitable sample pre-treatment
especially for a quantitative extraction of bacteria and viruses
from solid foods, limited availability of appropriate controls to
evaluate the effectiveness of the analytical procedure, the
current inability of molecular methods to distinguish DNA
from viable cells and DNA from dead or non-cultivable cells,
and the slow progress of international harmonisation and
E. Kudirkiene
Department of Food Safety and Quality, Lithuanian University of
Health Sciences, Veterinary Academy Tils Str. 18,
LT-47181 Kaunas, Lithuania
A. Hohl
Department of Food Science and Technology, Institute of Food
Science, University of Natural Resources and Life Sciences,
Muthgasse 18, Vienna, Austria
D. Horvatek Tomic : E. Prukner-Radovcic
Department of Poultry Diseases with Clinic, Faculty of Veterinary
Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb,
Croatia
K. A. To
School of Biotechnology and Food Technology, Hanoi University of
Science and Technology, No. 1, Dai Co Viet, Hanoi, Vietnam
M. Wagner
Department for Farm Animals and Veterinary Public Health, Institute
for Milk Hygiene, Milk Technology and Food Science, University
for Veterinary Medicine, Veterinrplatz 1, 1210 Vienna, Austria
256
standardisation, which limit full acceptance of PCR-based
methods in food control. The aim of this review is to describe
the context and the prospects of PCR-based methods, as well
as trends in research and development aimed at solving the
next decade challenges in order to achieve full integration of
molecular methods in food safety control.
Keywords Food safety . Rapid methods . Quality control .
PCR
Introduction
Microbiological analysis is an integral part of technological
food safety quality control and monitoring systems (Fig. 1).
Microbiological testing of foodstuffs complements activities
based on implementation and application of the concept of
Hazard Analysis and Critical Control Points (HACCP). In
addition to at-plant-implemented self-control programs, defi-
nition and application of microbiological criteria (MC) are a
salient feature in food legislation and in providing inputs to
microbiological risk assessment (MRA) (CodexAlimentarius
2012; Hoorfar 2011).
The advances in MRA nowadays extrapolated as quantita-
tive microbial risk assessment (QMRA), and the translation of
QMRA outcomes into risk management frameworks has led
to the establishment of a series of additional food safety risk
management metrics such as Food Safety Objectives (FSOs),
Performance Objectives (POs) and Performance Criteria (PC).
Where QMRA models are available or these metrics have
been elaborated, those allow the establishment of a more
direct relationship between MC and public health outcomes
(Manfreda and De Cesare 2014).
Fig. 1 The main purposes of
microbiological criteria according
to Proposed draft revision of the
principles for the establishment
and application of
microbiological criteria for foods
Codex Committee on Food
Hygiene Forty-fourth Session
New Orleans, United States of
America, 1216 November 2012
Food Anal. Methods (2015) 8:255271
Whereas FSO is defined as the maximum frequency and/
or concentration of a (microbial) hazard in a food at the time of
consumption that provides the appropriate level of protection
of health protection, PO defines the maximum frequency
and/or concentration of a microbial hazard at a specific step
of food chain. The concentration of microbial hazard should
be lower than the defined FSO if micro-organisms can prolif-
erate in the food product during its shelf life. As a direct
consequence of the introduction of the concept of FSO/PO,
the impact of assessing MC will increase, since these provide
objective means that PO or PC (or FSO) are being met at the
different steps of food chain. POs unlike FSOs are intended to
be easily measurable and verifiable.
FSO provides a recognised acceptable tolerance in
microbiological hazards, and it also represents moving
away from the precautionary principle (zero tolerance
concept) that was widely associated with microbiologi-
cal end-product testing. A statement illustrating this
change in paradigm is moving test activities from the
point of sale to the point of risk. These consider-
ations, however, imply a move from qualitative data
generation based on detection of an organism to a
quantitative approach. In addition, determination of PO
at different steps of food chain requires the definition of
a quantitative MC that might be sometime rather low
and should encompass the quantification of infective
micro-organisms only.
This new risk-based metrics approach to food safety moves
the MC from qualitative to quantitative, and the microbiolog-
ical methods have the main role to evaluate the effectiveness
of the HACCP systems by a quantitative approach. Prolonged
analysis times typical of classical culture-based microbiolog-
ical methods are incompatible with the HACCP systems.
Food Anal. Methods (2015) 8:255271
These systems could benefit from the application of the so-
called rapid methods, such as molecularbiological methods
(Amagliani et al. 2012). The development of rapid, cost-
effective and automated methods for the determination of
pathogenic bacteria, integrated with preventive strategies such
as HACCP, can significantly improve safety throughout the
food chain (Hofstra et al. 1994; Rodriguez-Lazaro et al. 2014).
These considerations served for discussions in an expert group
on rapid novel technologies, standardisation and
harmonisation of the European project safe food for
Europe (acronym FoodSeg). The aim of this review is to
report the discussion outcome concerning the need for future
research activities at European level, in order to reduce or
eliminate the drawbacks of molecular methods and to better
integrate the use in food safety control.
Food Safety and Food Hygiene Criteria
The Regulation (EC) No. 2073/2005 and following amend-
ments establish two types of criteria and require that food
business operators take corrective action when these criteria
are not met:
1. Food safety criteria should be used to assess the safety of a
product or batch of foodstuffs.
2. Process hygiene criteria should be used to ensure that the
production processes are operating properly.
Food safety criteria are applicable throughout the shelf life
of foodstuffs placed on the market. These criteria are
established for micro-organisms (usually pathogenic micro-
organisms), their toxins or metabolites in various food com-
modities, e.g. Listeria monocytogenes in ready-to-eat foods,
Salmonella in different foods of animal origin and vegetables,
Staphylococcal enterotoxin in certain cheeses, milk powder
and whey powder, Cronobacter spp. in infant formula foods
and histamine in fish. If a food safety criterion is not met, this
usually means that the food business operator will not be able
to place the foodstuff on the market or will need to remove the
food from the market (as stated in European Commission
Regulation 178/2002 laying down general food safety require-
ments). The food business operator has furthermore to under-
take steps that ensure that future production meets the
criterion.
Process hygiene criteria are used to assess the correct
functioning of production processes with respect to hygiene.
They are applicable to foodstuffs both during and at the end of
the manufacturing process but before the commercialisation
of the food products. Process hygiene criteria of a production
process are usually evaluated using indicator micro-
organisms, which provide information on the hygienic status
257
of food production in the food chain and indicate the
following:
& The level of biological and faecal contamination (indirect-
lythe level of risk from intestinal pathogens)
& The effect of technological processes
& Prognosis for the future spoilage of products stored
improperly
& Epidemiological risks of potentially pathogenic micro-
organisms
Specific bacterial indicators are available for defined types
of f ood p rodu cts and includ e Escherichia c oli,
Enterobacteriaceae, staphylococci and enterococci (either
staphylococci and enterococci, or Staphylococcus sp. and
Enterococcus sp.) (Kornacki and Johnson 2001). If a process
hygiene criterion is not met, the product can be placed on the
market, but the food business operator must review the pro-
duction processes and improve process hygiene to ensure that
future production will meet the criteria.
Food Safety and Food Analytics
The current legislation urges the food business operator
(FBO) to take the responsibility for the production of
safe food. FBOs establish self-control systems and
HACCP concepts as backbones to reach this goal. It is
essential to understand that the majority of food samples
are nowadays tested at FBO level, whereas the number
of food samples tested by public health authorities is
decreasing, in many cases due to cost-saving programs.
In Austria, the public health sectors test around 35,000
samples a year, which represent a small number com-
pared to the samples tested at FBOs within in-house
routines. The change in the legal framework segregated
food testing into applications used at food business
operations and those used by control authorities for
food control. FBO, however, can only take over this
responsibility if a sample can be analysed within the
time the food is stored at a food enterprise (FE).
Traditional microbiological methods are often consid-
ered to be unsuitable for evaluating food safety criteria
at FEs due to their long duration, which may lead to a
situation that the product is already supplied, or even
sold, before data on its safety status are available. As a
consequence, most quality managers ensuring food safe-
ty at food enterprises have moved into using rapid
technologies, whereas the public health authorities still
work with culture-dependent technology that is mostly
standardised by international standardisation bodies such
as the European Committee for Standardization (Comit
Europen de Normalisation (CEN)) or International
258
Organization for Standardization (ISO). Rapid, sensitive
and accurate detection methods are necessary to be
integrated with HACCP to improve safety of products.
Therefore, research efforts devoted to the development
of proprietary techniques (PTs) are still ongoing, and the
current literature contains many references to that ex-
tent. A PT measures the same analyte as the corre-
sponding reference (classical microbiological) method
and facilitates response through simple performance
and/or automation and better analytical performance (ac-
curacy, sensitivity, specificity). If the time parameter is
prioritised, the term rapid methods instead of PT is
often preferred.
Immunological and molecular biological nucleic acid-
based methods are currently the best examples of PT currently
implemented into food testing. This review focuses in partic-
ular on polymerase chain reaction (PCR)-based technologies
since immunological test was successfully introduced to food
testing in the previous decade and not much progress with
regard to the basic analytical principle that was achieved from
thereon. The introduction of amplification techniques (PCR
and real-time PCR) in microbial diagnostics has been
established in research laboratories as a valuable alternative
to traditional detection methods. Compared to immunological
methods, PCR-based technologies have two essential advan-
tages: (i) the biochemical principle of in vitro DNA amplifi-
cation, since DNA is a suitable analyte in every organism and
thousands of different organisms can be tested by very small
changes in the analytical format; and (ii) the ability to quantify
the analyte. Speed, excellent selectivity, specificity, sensitivity
and potential for automation are further important advantages
of amplification techniques. These advantages compared to
traditional detection methods might well encourage end-users
to adopt amplification techniques in routine testing for food-
borne pathogens (Rodriguez-Lazaro and Hernandez 2013).
Some previously identified obstacles of implementation of
molecular methods have been already overcome. The robust-
ness of PCR-based methods has been improved by developing
real-time PCR assays, and equipment and biochemical re-
agents have become much more affordable (Rodriguez-
Lazaro et al. 2013). Unfortunately, PCR-based methods still
suffer from certain drawbacks that hamper their full integra-
tion to food safety control, and those are the following:
& Lack of specific pre-treatments that allow quantitative
extraction of micro-organisms from foods, in particular
solid foods
& Inability of molecular method to distinguish DNA from
viable and dead cells
& Lack of definition of appropriate controls to evaluate the
effectiveness of the analytical procedure
& Slow progress of the international harmonisation and
standardisation of molecular methods
Food Anal. Methods (2015) 8:255271
Molecular Methods in Food Analysis
At the beginning of the second millennium, molecular
methods were considered not suitable for routine testing of
food products, because they looked good and worked well
only if used in research laboratories with skilful technicians.
However, in the last decade, nucleic acid-based methods
gradually started to replace or complement the culture-based
methods in food control (Kuchta et al. 2014) and became
extensively utilised as alternative rapid methods for detecting
pathogenic bacteria. For several pathogens, complete detec-
tion procedures of different commercial and non-patented
methods, including enrichment, were developed and validated
(Rodriguez-Lazaro and Hernandez 2013; Rodrguez-Lzaro
et al. 2007).
In this context, several international studies were per-
formed in particular in the Framework of different EU
founded projects. Particularly, an interlaboratory trial, involv-
ing 15 laboratories from 13 European countries, was conduct-
ed to evaluate the performance of a non-proprietary gel-based
PCR method for the detection of Salmonella on artificially
contaminated chicken rinse and pig swab samples (Malorny
et al. 2004).
Another assay for the detection of L. monocytogenes was
evaluated in a collaborative trial involving 12 European lab-
oratories (DAgostino et al. 2004).
The performance of a PCR-based method for the detection
of E. coli O157, previously validated on DNA extracted from
pure cultures, was evaluated on spiked cattle swabs in an
interlaboratory trial, including 12 participating laboratories
from 11 European countries (Abdulmawjood et al. 2004).
In addition, an ISO 6579 compatible enrichment coupled to
an easy and inexpensive DNA extraction and a consolidated
real-time PCR assay for detecting Salmonella in pork meat
was evaluated in a ring trial performed in 13 laboratories from
seven European countries (Delibato et al. 2014).
Twelve laboratories from six European countries partici-
pated in a ring trial for evaluation of a real-time PCR-based
method for the detection of L. monocytogenes in soft cheese as
food model since representing a difficult matrix for bacterial
DNA extraction and real-time PCR amplification
(Gianfranceschi et al. 2014).
Also, a ring trial study for evaluating the performance of a
real-time PCR-based method for the detection of botulinum
neurotoxin producing clostridia in food was performed in four
different European laboratories in Italy, France, the
Netherlands and Sweden using 47 strains and 30 clinical and
food samples linked to botulism cases (Fenicia et al. 2011). In
addition, this ring trail was performed in order to improve
quality assurance also for the detection of deliberate contam-
ination in the food chain (Knutsson et al. 2011).
A multi-centre collaborative trial involving eight laborato-
ries in five European countries (two laboratories in France,
Food Anal. Methods (2015) 8:255271
Italy and the Netherlands and one laboratory in Denmark and
Sweden) was also performed in order to validate a real-time
PCR-based method for the detection of Clostridium botulinum
types C and D and their mosaic variants CD and DC
(Woudstra et al. 2013).
Differently from the detection of bacteria in food where
rapid method was used alternatively to the classical cultural
method, real-time PCR is the exclusive method for the detec-
tion of viruses in food.
Norovirus do not grow in any cell culture-based detection
system (Duizer et al. 2004), while human wild-type hepatitis
A virus (HAV), even if rarely can grow in a cell culture, is
genetically unstable and requires several weeks to months in
culture before it can be detected (Konduru and Kaplan 2006).
Consequently, a real-time PCR was raised as an elective
method for the detection of the presence of these viruses in
food, water or environmental samples.
Since the presence of enteric viruses in food samples is
widely recognised (Koopmans et al. 2002), in particular in
mussels (Suffredini et al. 2014; Pavoni et al. 2013) and in
fresh product as frozen soft fruits (Rizzo et al. 2014), different
protocols were proposed for the detection of HAV (Sanchez
et al. 2007) and noroviruses (Stals et al. 2013) in food using
real-time PCR. Recently, a method for the detection of
noroviruses was validated on naturally contaminated fresh
produce samples (El-Senousy et al. 2013).
Advanced Sample Treatment and Nucleic Acid Extraction
of Bacteria and Viruses
The pre-PCR processing step, including sample preparation
and nucleic acid extraction, is one of the most critical steps in
molecular biological analysis. It has considerable influence on
reproducibility of the analysis especially when quantitative
detection of a specific pathogen is needed. Most of the mo-
lecular biological methods used are in vitro methods, which
work perfectly in a standardised analytical environment that is
practically met only under perfect laboratory conditions. The
theoretical or in vitro performance of these methods is
challenged when differences in analytical skills or sample-
dependent inhomogeneity come into play. The sample prepa-
ration and DNA extraction steps should aim at minimizing
inhomogeneity through achievement of a certain level of
robustness and analyte quality that should be mainly
characterised by analyte purity and integrity. Moreover, for
quantitative purposes, the amount of analyte initially present
in the food sample should not be changed through the se-
quence of manipulations in the analytical chain. The holistic
view on all steps in the analytical chain is unequivocal. This is
of importance since extensive research on some steps has led
to an overwhelming number of papers (so more than 200
papers have been published on PCRs for Salmonella),
259
whereas other steps remained widely out of focus. The ana-
lytical chain is composed of sample preparation, nucleic acid
extraction and application of the amplification or detection
platform (Rossmanith and Wagner 2011). Unlike the clinical
microbiology that works on a limited number of types of
mostly liquid specimens, food samples are highly heteroge-
neous in composition and physical state. The nucleic acids
need to be purified from all other compounds that might
originate from the food and could influence the biochemical
kinetics of PCR. It has to be considered that pre-PCR process-
ing step is often not included in validation. Therefore, it is
suggested that standardised PCR methods also include
methods for sample processing in order to overcome some
of the major problems associated with the sample by concen-
trating the target organism, reducing the presence of inhibitory
substances (collagen, polysaccharides, proteinases and calci-
um) and converting a heterogeneous sample into a homoge-
neous sample suitable for PCR (Postollec et al. 2011; Malorny
et al. 2003; Hoorfar et al. 2004a; Radstrom et al. 2004;
Radstrom et al. 2008). Clearly, it is required that PCR is even
more adapted for field use, which is quite far from perfect
laboratory conditions.
The procedures of sample preparation have gradually re-
ceived increasing attention from both academia as well as
industry, mirrored by an increase in publications as well as
applications of such methods (Brehm-Stecher et al. 2009).
However, so far, the topic remains a challenge for research
as there are many prerequisites for efficient sample prepara-
tion methods (Rossmanith and Wagner 2011; Stevens and
Jaykus 2004; Rossmanith and Wagner 2010). The vast num-
ber of procedures used for this purpose indicates that this
challenge is still a subject of intensive research. Most methods
have drawbacks, such as insufficient size of the processed
sample, insufficient recovery, extensive labour requirements
or high costs. Particularly, promising approaches to overcome
sample-related problems are separation using coated magnetic
beads specific for the analyte, flotation or other physical
methods and the lysis of whole sample matrices
(Rossmanith et al. 2007).
Improved immunoseparation methods have become avail-
able for rapid identification of cultivable and non-cultivable
micro-organisms (Olsvik et al. 1994). The principles and
application of the method are simple but rely on antibodies
of suitable specificity under the conditions of use. Purified
antigens are typically biotinylated and bound to streptavidin-
coated paramagnetic particles. Immunomagnetic beads (IMB)
have a potential to become convenient tools also for multi-
pathogen detection. Tu et al. (2011) used streptavidin-coated
magnetic beads conjugated with biotinylated capture antibod-
ies to separate both E. coli O157:H7 and Salmonella
Typhimurium in a culture system. These were multi-
pathogen capture IMB (IMB-M). The efficacy of these beads
was investigated in both pure and mixed culture suspensions,
260
as well as in inoculated spinach and ground beef. It was found
that IMB-M were just as effective as the mixture of IMB
against E. coli and Salmonella in capturing cells of both
organisms. Using this approach, it was possible to detect
1 CFU/g of E. coli and 100 CFU/g of Salmonella in a mixed
culture, after 6-h enrichment at 37 C.
Another approach combines cell capture using magnetic
particles (MP) with multiplex PCR, offering an efficient,
rapid, sensitive and inexpensive alternative for the routine
detection of food-borne pathogenic bacteria in food and
stools. A recently published study demonstrated a novel mag-
netic capturemultiplex PCR assay for the simultaneous de-
tection of E. coli O157:H7, Salmonella and Shigella in stool
and food samples (Zuo et al. 2011).
Innovative sample preparation strategies should also over-
come the problem that food-borne pathogens are usually
present in food commodities at very low numbers.
Therefore, novel sample preparation technologies process
higher sample volumes. A re-circulating automatic
immunoseparator Pathatrix Auto has recently gained
AOAC-RI approval for its Salmonella, Listeria and E. coli
O157 detection product range. This technology provides
shortening of the time necessary for enrichment prior to
PCR. The system is able to concentrate bacterial cells from
up to 60 ml of pre-enriched food samples, producing a con-
centrate of 100 l. The concentrate is processed by cell lysis
and DNA extraction and further subjected to real-time PCR. A
pooling option of the system is available for applications
where negative results prevail (Shields et al. 2012;
Papafragkou et al. 2008; Lau et al. 2012; Zhao et al. 2012).
This re-circulating immune separation technology has been
also applied to the concentration of HAV from food samples
(Papafragkou et al. 2008). The hypothesis on the working
mechanism is that cationically charged magnetic particles
could be used in conjunction with this magnetic capture
system for the concentration and purification of the virus from
food matrices. The separation of the viral agent is facilitated
by binding the negatively charged proteins of the virus capsid
to the positively charged magnetic particles (Papafragkou
et al. 2008). A major advantage of the system is that large
volumes can be analysed (25 g of food, plus 225 ml of buffer),
and the resulting sample is concentrated up to 500-fold.
An inexpensive sample preparation method for the separa-
tion of gram-positive micro-organisms from various food
matrices and blood was recently developed. This procedure,
called matrix lysis, involves solubilisation of the food ma-
trix followed by concentration of intact bacteria through cen-
trifugation to detect <10 CFU/g target micro-organisms, while
free DNA is eliminated by 5 log units (Mayrl et al. 2009;
Rossmanith et al. 2007; Aprodu et al. 2011). This procedure is
friendly, specific and rapid, and high amounts of food samples
can be processed. So far, four different buffer systems have
been successfully introduced, two of them exclusively for
Food Anal. Methods (2015) 8:255271
real-time PCR quantification, while the latest two buffer sys-
tems, based on a novel chemical substance class called ionic
liquids (IL) or on MgCl2, also allowed for the application of
microbiological methods after matrix lysis (Mayrl et al. 2009;
Mester et al. 2010b; Rossmanith et al. 2011; Mester et al.
2010a).
Nucleic acid purification methods should be characterised
by a high efficiency (almost all DNA from the target cells
present is harvestable), purity (low amounts of other macro-
molecules are co-extracted) and integrity (the nucleic acid
remains undegraded through the chemical and sometimes
mechanical procedures applied). Nucleic acid extraction often
utilises cell wall lysing chemistry (such as guanidine thiocy-
anate to denature viral and bacterial coat proteins) in combi-
nation with resins to bind the released nucleic acid, which is
then purified through successive washing steps before final
elution in a small volume (EFSA 2011). Nucleic acid extrac-
tion should work efficiently either on DNA or RNA.
Particularly with enteric RNA viruses, for the detection of
which a reverse transcription stage is necessary, the capacity
of an extraction method to obtain a nucleic acid sample as pure
as possible is a particularly important point. Indeed, the high
susceptibility of reverse transcriptase to inhibitory substances
is a major limiting factor in such methods (Wilde et al. 1990).
A lot of methods have been proposed for extracting viral RNA
and simultaneously reducing the levels of inhibitors (Butot
et al. 2007b). For bivalve molluscs, dissected digestive diver-
ticulum (digestive gland) is used as the starting material with
further enzymatic digestion using proteinase K (Jothikumar
et al. 2005). For food contact materials, swabbing is
employed, followed by elution into buffer (Scherer et al.
2008). For molecular biological analysis of water, including
bottled water, the established methods usually involve adsorp-
tion of viruses on a positively or negatively charged mem-
brane, and then the adsorbed viruses are eluted and concen-
trated by ultrafiltration (Butot et al. 2007a). In order to eval-
uate the efficiency of the entire analytical procedures to con-
centrate enteric viruses in bottled waters, an ultrafiltration
method using charged filtration membrane was recently eval-
uated step by step. The results showed that a considerable
number of virus particles passed through the pores of the
membranes instead of being trapped by the electrostatic
charges (Di Pasquale et al. 2010). A new method able to
extract all the virus particles trapped by the membrane has
been developed (Perelle et al. 2009) and validated in a
European ring trial (Schultz et al. 2011). Recently, improve-
ment in the release rate of virus particles from the membrane
by the use of ultrasound prior to ultrafiltration was proposed
for detecting enteric viruses from different types of bottled
waters (Butot et al. 2013).
Detection of viruses in fruits and vegetables starts with the
elution of the virus particles from the surface of the product
(Sadovski et al. 1978) because it is assumed that naturally
Food Anal. Methods (2015) 8:255271
contaminated samples carry virus particles only on the sur-
face. However, HAV particles were found also trapped inside
growing green onions probably after being taken up intracel-
lularly through the roots (Chancellor et al. 2006). In lettuce,
the viral contamination of the plants via roots cannot be
excluded but is apparently not an important transmission route
for viruses (Urbanucci et al. 2009).
Filtration through large porosity filters, previously treated
to reduce the adsorption of viruses, has also been used to
remove food particles. This procedure was very useful also
to concentrate viruses from raspberries and frozen fruits; in
this case, pectinase has to be added to prevent jelly formation
in the eluate (Croci et al. 2008).
Immunochemical methods have also been applied to the
separation of viruses from food samples (Bidawid et al. 2000;
Kobayashi et al. 2004; Shan et al. 2005; Tian et al. 2006; Tian
and Mandrell 2006). However, the development of an
immunoconcentration procedure for noroviruses has encoun-
tered difficulties with obtaining antibodies and with their
variability at the capsid level.
The discovery of human histo-blood group antigens
(HBGA) on cells of the human gastrointestinal tract as
norovirus receptors has been used for developing
immunoconcentration procedures for selective binding of
NoV in faecal and food samples (Tan and Jiang 2005;
Huang et al. 2005; Le Guyader et al. 2006).
Detection of Viable Cells or Infective Viruses
Traditionally, bacteria are considered viable when they can be
cultured. This concept is not applicable when the detection of
bacteria is performed by molecular methods, since the detec-
tion is focused on bacterial DNA. Conclusively, an important
drawback of nucleic acid-based methods is their inability to
distinguish DNA from viable cells and DNA from dead (or
non-cultivable) cells. Different techniques were developed to
detect only viable bacterial cells by molecular methods, such
as the detection of messenger RNA (mRNA) and the use of
DNA-intercalating dyes (propidium monoazide (PMA) and
ethidium monoazide (EMA)).
An appropriate viability criterion is the active metabolism
of cells (Nocker and Camper 2009). Detection of mRNA by
molecular methods has been studied extensively as it is
strongly believed that its presence correlates with cell viability
(Gonzalez-Escalona et al. 2009; Coutard et al. 2005; Hellyer
et al. 1999; Liu et al. 2010). Different studies demonstrated
that targeting mRNA may reduce the possibility of false
positive results at determination of viable cells, since the
half-life of bacterial mRNA (hours) is much shorter than that
of DNA (days or months). In contrast, other studies showed
that both DNA and mRNA may persist in a detectable form
for many hours after cell death (Birch et al. 2001) and
261
demonstrated a potentially poor correlation between mRNA
detection and cell viability. Practical drawbacks are that
amplifiable template RNA may be difficult to extract from
foods due to the presence of inhibitors, such as fat, proteins
and components from blood cells, the method being efficient
only if all the bacterial background DNA is removed enzy-
matically by DNase treatment.
In fact, the removal of DNA contamination in the RNA
extract by DNase treatment has to be confirmed by PCR
before the application of mRNA-based real-time (RT)-PCR
in protocols for the detection of living cells. Repeated DNase
treatment may sometimes be necessary, since some bacterial
DNA may be more resistant to DNase treatment (Kobayashi
et al. 2009). This illustrates that the process is technically
rather complicated and more time-consuming than DNA-
based PCR, which limits the application of the mRNA-based
methods in routine laboratory testing.
Another approach to a selective detection of living micro-
bial cells is the use of EMA or PMA in combination with
PCR-based molecular diagnostic techniques. This approach
was recently used in several studies and was reported to be an
easier-to-use alternative to mRNA detection. The use of EMA
or PMA was effectively evaluated in different bacteria
(Josefsen et al. 2010; Wolffs et al. 2001) and viruses
(Parshionikar et al. 2010). PMA and EMA are two DNA-
intercalating dyes that penetrate only to dead cells with com-
promised cell membrane integrity but not to viable cells with
intact cell membranes. The presence of an azide group is
believed to permit cross-linking of the dye to DNA after
exposure to strong visible light. When exposed to light, pho-
tolysis of EMA and PMA causes the azide group to be
converted to a nitrene radical, which covalently binds to any
carbon moiety in proximity, including both extracellular and
double-stranded DNA contained in cells with compromised
membranes, forming a carbonnitrogen bond. This covalent
linking inhibits amplification by PCR. DNA with bound EMA
is insoluble and can be removed with cell debris during the
DNA extraction process (Nocker et al. 2007).
However, it has been reported that EMA can also, to some
extent, penetrate viable bacterial cells and there covalently
cross-link with DNA during photolysis (Rudi et al. 2005;
Nogva et al. 2003). This may result in the loss of some
percentage of the genomic DNA of viable cells (Nocker
et al. 2006). In Campylobacter, recent research has shown
that EMA has a toxic effect thus contributing to the load of
dead cells (Flekna et al. 2007). Recently, Minami et al. (2010)
proposed treating bacteria before real-time PCR with a con-
centration gradient of EMA. This approach may increase live
and dead distinction ability to assure that EMA destroyed only
DNA derived from dead cells. This newly developed low-
dose double-treated EMA-PCR was demonstrated to be very
effective for viable Cronobacter sakazakii detection in pow-
dered infant formula (PIF) (Minami et al. 2012). Interaction of
262
monoazide compounds with bacterial DNA is complex, and it
may depend on concentration of the dye, concentration of free
DNA, as well as on numbers of damaged and viable cells
present in the sample.
Another way to improve this technique may be based on
the finding that long-amplicon PCR is more sensitive to
monoazide treatment of the template DNA. Long-amplicon
PCR would require less monoazide treatment to block DNA
from dead cells and thus reduce the loss in the detection of live
cells (Soejima et al. 2011).
Further research should focus on the influx and efflux of
EMA or PMA to cells of different bacterial species and strains
at different conditions of incubation and the efficiency of
cross-linking via photo-activation to free DNA, DNA within
dead and damaged cells and DNA within viable cells (Flekna
et al. 2007).
A physical separation approach that can be used to separate
viable and dead cells is flotation, a technique that utilises their
different densities. The cell densities of 12 different bacterial
strains, determined by centrifugation using a step density
gradient of Percoll, were between a very strictly defined range
(1.031 and 1.120 g/ml) (Fukushima et al. 2007). This range of
densities allowed the separation of living bacterial cells in
stationary phase from the food matrix and from dead bacterial
cells.
The benefits of density gradient centrifugation as a sample
pre-treatment method are well established and include (i) the
possibility of separating biological matrix particles and cells
of micro-organisms with different buoyant densities, (ii) main-
taining cell viability, which allows isolation and analysis of
the micro-organisms, and (iii) speed and easy handling. To
date, this technique has been used only for the detection of
Yersinia enterocolitica in pork meat juice and for quantifica-
tion of Campylobacter in chicken rinse samples (Wolffs et al.
2004; Wolffs et al. 2005). Separation of food-borne pathogens
is thought to be more feasible in liquid samples rather than in
solid ones, and flotation may only be used on liquid samples.
The aforementioned concept of matrix lysis does not impair
cell viability when using ionic liquids and can be used both
with PCR or conventional isolate identification (Mester et al.
2010b). This concept was further researched by DUrso et al.
(2009), who described a new filtration-based method for the
detection of viable Listeria and Salmonella cells in food
samples (DUrso et al. 2009). In general, analytical problems
are caused by bacteria arrested in a viable but not cultivable
(VBNC) state mostly after increased stress. VBNC may cause
a deviation between quantities determined by classical micro-
biology (as CFU) and data obtained by culture-independent,
molecular quantification (Reichert-Schwillinsky et al. 2009).
In order to differentiate viable from non-viable cells, not
only in food where the bacteria are present on surface but also
in the mass of the food matrix, the use of a short enrichment
step has been also proposed (Kramer et al. 2011). The method
Food Anal. Methods (2015) 8:255271
combines a short (8 h) non-selective pre-enrichment step able
to produce only viable cells in log-phase and a real-time PCR
to evaluate quantitatively the number of living bacteria. This
approach was used to enumerate Salmonella in different egg
products (Jakociune et al. 2013) and to detect verocytoxigenic
E. coli (VTEC) in milk (Mancusi and Trevisani 2014).
However, the use of any non-selective cultivation may
lead, in certain food matrices and in certain mixed cultures
of micro-organisms, to a relative suppression of minor com-
ponents of the microflora and thus affect the results of the
downstream analysis (Krascsenicsova et al. 2006).
What is true for bacteria constitutes an analytical problem
in virus detection as well. The burden of infective enteric
viruses in a food is crucial to evaluate the real risk for human
health (Kim et al. 2011; Kim and Ko 2012). Unfortunately,
molecular methods are unable to distinguish between infec-
tious and non-infectious viruses, where upon the latter usually
consist of defective virus particles, containing different
amounts of intact or degraded viral RNA (Knight et al.
2012). It was demonstrated that viral genomes may persist in
the environment for a longer time than the viral infectious
particles (Richards 1999; Ogorzaly et al. 2010). For example,
the HAV genome was detectable by RT-PCR for 232 days,
while virus particles were detectable in cell culture for only
35 days. This suggests that the detection of the HAV genome
by RT-PCR is not a reliable indicator of the presence of a
viable virus (Koopmans et al. 2002).
Cell culture assay is the standard method for the detection
of viable human viruses (Nuanualsuwan et al. 2002), and the
use of a method integrating cell culture and PCR in order to
confirm the viability (infectivity) of the viruses proved effec-
tive (Hyeon et al. 2011; De Medici et al. 2001; Reynolds
2004). Unfortunately, the cell culture method is quite time-
consuming, as up to 2 weeks is necessary to confirm the
results for HAV, and it is not applicable to norovirus, which
cannot grow outside of the human host. Analogically to bac-
teria, the idea of using EMA or PMA could be a step forward
to identify infective enteric viruses, since the absence of a cell
culture system for some food-borne viruses hampers the de-
termination of infectivity of viruses when isolated from con-
taminated foods. Again, the combination of dye and the target
organism seems to be of great importance, as demonstrated in
a study in which EMA failed to distinguish between viable
and non-viable virions of avian influenza virus (AIV) (Graiver
et al. 2010). The AIV envelope is similar but still different
compared to a bacterial cell membrane, and it is possible that
EMA is unable to penetrate into membrane-compromised
AIV virions.
Sanchez et al. (2013) demonstrated that PMA treatment
prior to RT-quantitative PCR (qPCR) detection was a prom-
ising method for assessing HAV infectivity. The use of PMA
or EMA in conjunction with three surfactants (IGEPAL CA-
630, Tween 20, Triton X-100) prior to RT-qPCR was also
Food Anal. Methods (2015) 8:255271
shown to be effective to quantify the infectious particles of
HAV and of two strains of RV (SA11 and Wa), after heat
treatment (Coudray-Meunier et al. 2013).
Owing to the importance of the viral capsid in infectivity,
the measure of the capsid integrity or virolysis may repre-
sent an alternative marker for virus infectivity in the absence
of a cultural model. Capsid integrity has been investigated
using combined proteinase K and RNase treatment, in order to
correlate PCR data with infectivity (Nuanualsuwan and Cliver
2002). However, this treatment was difficult to control be-
cause it depends on relative activities of individual enzymes,
making the results difficult to reproduce. Assuming that non-
intact capsids would not be able to bind to appropriate recep-
tors, it was proposed to utilise the binding affinity of virus
particles to their natural receptors or other binding ligands
(Nuanualsuwan and Cliver 2003; Ogorzaly et al. 2013). For
instance, ligands, such as human histo-blood group antigens,
demonstrated a high grade of affinity to noroviruses and thus
have been considered as an alternative to antibodies (Morton
et al. 2009).
Appropriate PCR Control
PCR-based detection of micro-organisms in food is a compar-
atively complicated process involving several steps, all of
them being possible sources for errors. Those encompass
improper sample preparation (enrichment, cell lysis, DNA
extraction, removal of inhibitors) or failure of PCR (compo-
sition of reaction mixture, performance of the PCR cycler). In
order to be able to control the PCR-based analysis, various
types of controls are recommended for use.
The correct performance of the reaction mixture can be
checked by the external (positive) control. However, it is more
important to control if PCR was successful in each tube and in
each position of the cycler block. For this purpose, an internal
amplification control (IAC) can be used, which is added to
each PCR tube. Many different formats have been described
so far, for example various plasmids or bacteriophage DNA
may be used as a template (Fricker et al. 2007). This template
is added at a low concentration, and it is detected by its own
primers/probe designed to work at the same PCR temperature
programme. Theoretically, the addition of an exogenous IAC
should not affect the main analytical reaction. However, this
inertness has to be checked for each application (main primer/
probe system, reaction mixture composition, temperature pro-
gramme) so as to avoid excessive primerdimer formation.
Another technical alternative to IAC is the use of a specific
template that can be amplified with the same primers, so-
called mimic, in the same reaction conditions (Thisted
Lambertz et al. 1998). In this setting, primers compete for
the main target and for the control target. The products of the
main PCR and the control PCR are distinguished based on the
263
size of the amplified DNA fragment or using fluorogenic
probes labelled with different dyes. An advantage of this
approach is a reduction in the number of types of oligonucle-
otides present in PCR, which reduces the risk of their unwant-
ed interaction. A drawback is that each diagnostic primer/
probe system requires a new mimic molecule.
For the same purpose, an endogenous control, external or
internal, can be used. This is a separate primer/probe system
targeting some universal DNA sequence present in any pro-
karyotic (Greisen et al. 1994) or eukaryotic DNA (Brezna
et al. 2006). A preferred format of this type of control is
external, so as to avoid the situation that a strong control
signal affects the signal from the main reaction.
A no-template control as a negative control should be
included in each PCR-based method for food control. This is
prepared in a separate tube and contains all PCR components
with the exception of any DNA template. The results of this
reaction should be always negative. An accidental positive
result would indicate contamination of working solutions,
tubes or pipette tips by DNA.
The ideal type of control would, however, be the one that
controls the entire analytical procedure. This might be, for
example, a freeze-dried culture that would be added to the
food sample prior to enrichment, would pass all steps of the
analysis (enrichment, lysis, DNA extraction, PCR) and then
would be selectively detected by PCR along with the target.
The requirements suggest that it probably would be a micro-
organism that is related to the target, cultures of which are able
to grow in all enrichment media, but not faster than the target.
Either well-defined wild or specifically designed engineered
micro-organisms may suit this purpose (Rossmanith et al.
2011). Quantitative levels of such entire-process internal con-
trol to be applied as well as their physiological state (e.g.
mildly stressed) should be well studied so as to avoid the
suppression of growth of cultures of the target micro-
organism. Phenotypic as well as genetic stability of control
micro-organisms should be studied, in particular if engineered
micro-organisms are intended to be used for this purpose.
The use of an appropriate process control has been also
debated for the evaluation of the rate of recovery in the quanti-
tative detection of viruses in different food matrices. Particularly,
during the development of the method by the Tag 4 of CEN
working group, a genetically modified strain of Mengo virus
was successfully utilised as a process control (Costafreda et al.
2006). The use of feline calicivirus was also proposed as an
alternative process control (Di Pasquale et al. 2009).
Standardisation and Validation of Molecular Method
Protocols
According to the official definition of ISO, the term standard
describes a document established by consensus and approved
264
by recognised body that provides, for common and repeated
use, guidelines or characteristics for activities or their results,
aimed at the achievement of the optimum degree of order in a
given context(ISO/IEC 2004). A standard is of common
interest and reflects the concerns of all stakeholders involved
by implementing the largest consensus and the broadest ap-
plicability in food laboratories worldwide. Listing or
recommending commercial products in standard protocols is
avoided as much as possible. Standards can be issued at the
national level (national standardisation bodies), the regional
level (e.g. CEN) or the international level (ISO). Most stan-
dards in food analysis are developed by CEN/TC275/WG6
(CEN Working Group 6 Microbial contaminants of
Technical Committee 275 Food AnalysisHorizontal
methods of CEN) on the European level and by ISO/TC 34/
SC 9 (ISO Sub-Committee 9 Microbiology of Technical
Committee 34 Food Products) on the international level. As
outlined in the Vienna Agreement, usually, standards devel-
oped by SC 9 are adopted also as CEN standards and vice
versa (Lombard and Leclercq 2009).
Standardisation of modern technologies such as molecular
methods can be achieved either by the development of specific
standards for novel technologies or by elaborating standards
laying out the general guidelines for applying PCR in food
analysis. Resolution No. 233 of the joint meetings of ISO/
TC34/SC9 and CEN/TC275/WG6 states that novel technolo-
gies can be introduced to a reference standard method if the
performance criteria of the reference method are not satisfying
or if the novel methodology targets not taxonomy (like the
conventional method) but e.g. pathogenicity. Furthermore,
resolution 233 stipulates that commercial products are not to
be mentioned in the standard but that their validity should be
checked against the standard reference method (Lombard and
Leclercq 2009). This idea of validating data produced by
culture-independent analysis against data of classical culture-
dependent microbiology was well described in a recent paper
where the authors suggested a novel strategy on how to
validate molecular data by system analysis (Rossmanith and
Wagner 2011).
Use of PCR in food microbiology is covered by the Tag 3
expert committee of CEN/TC/275/WG6. To date, only few
methods based on the use of molecular techniques are
harmonised and standardised by ISO/CEN. The majority of
published ISO/CEN norms deal with the general aspects of
molecular methods, while other norms are already at draft
level or in discussion (Table 1). In 2004, CEN initiated the
development of a standard method for the detection of
norovirus and HAV in foodstuffs based on PCR (Lees 2010),
and this norm was recently published (Table 1). Specific
norms are missing for molecular detection of the most impor-
tant food-borne micro-organisms.
Recently, norm ISO/TS 17919:2013 for the detection of
botulinum type A, B, E and F neurotoxin-producing clostridia
Food Anal. Methods (2015) 8:255271
using PCR was published. Some of the methods proposed in
the norm are evaluated in European collaborative works (De
Medici et al. 2009; Lindstrom et al. 2001; Fach et al. 2009).
Validation protocols on the national and international level
have been developed by many bodies. On the international
level, one of the most recognised and hence important proto-
cols is the ISO standard 16140:2003 Microbiology of food
and animal feeding stuffsProtocol for the validation of
alternative methods. This standard outlines general principles
and the technical protocols for the validation of alternative
microbiological methods. Details about method comparison
studies and interlaboratory studies are stated here for both
qualitative and quantitative methods. An overview of the
required parameters is given in Table 2.
Another key organisation in validation and standardisation
of methods for microbiological testing of food is the
Association of Official Analytical Chemists (AOAC)
International. AOAC currently run two validation
programmes: the Official Methods of Analysis (OMA) and
the Performance Tested Methods Programme (PTM). A third
programme, the peer-verified methods, was disbanded in July
2008. AOAC recently added a third programme of First
Action methods to facilitate rapid adoption of new methods,
which includes also microbiological methods based on PCR.
According to the MoniQA European project working
group (www.moniqa.eu) when comparing the protocols of
the main validation organisations, there is a clear need for
uniformity in different areas (Table 3). One such area is a
standard strain collection used for validating methods. For
example, AOAC specify that isolates should be obtained
from ATCC, while other validating organisations will accept
strains acquired from regional culture collections or in-
house collections. There is a wide variation in both the type
of enrichment methods and the primary enrichment conditions
employed in various validation studies. For example, with
some pathogens, different enrichment methods are chosen to
validate different methods, and different enrichment methods
can be used to validate the same methods with different food
matrices.
Certain changes would be also useful regarding the wide
variety of food matrix materials used for detection and enu-
meration in validation studies. Such variety may be useful at
assessing repeatability of the methods but not for comparing
methods between studies. Contra productivity of the current
approach may be illustrated by an example of molecular
detection of thermo-tolerant Campylobacter, for which an
unexpected matrix effect was observed, leading to underesti-
mation of the bacteria in milk (Rosenquist et al. 2007).
With molecular methods, where reproducibility, reliability
and accuracy may depend on a broad range of variables, there
is a need to precisely detail all relevant information when
conducting collaborative trials. For example, with PCR-
based methods, the type or brand of thermo-cycler (Schoder
Food Anal. Methods (2015) 8:255271 265

Table 1 The status of the standard references on use of molecular methods for detecting pathogenic micro-organisms in food and water

Standard reference Title Status

EN ISO 20837:2006
EN ISO 20838:2006
EN ISO 22118:2011
EN ISO 22119:2011
EN ISO 22174:2005
CEN ISO/TS 13136:2012
ISO/TS 15216-1:2013
ISO/TS 15216 -2:2013
ISO/TS 17919:2013
ISO/CD TS 18867
Microbiology of food and animal feeding stuffspolymerase chain reaction (PCR) for the detection of published
food-borne pathogensrequirements for sample preparation for qualitative detection
Microbiology of food and animal feeding stuffspolymerase chain reaction (PCR) for the detection of published
food-borne pathogensrequirements for amplification and detection for qualitative methods
Microbiology of food and animal feeding stuffspolymerase chain reaction (PCR) for the detection and published
quantification of food-borne pathogensperformance characteristics
Microbiology of food and animal feeding stuffsreal-time polymerase chain reaction (PCR) for the published
detection of food-borne pathogensgeneral requirements and definitions
Microbiology of food and animal feeding stuffspolymerase chain reaction (PCR) for the detection of published
food-borne pathogensgeneral requirements and definitions
Microbiology of food and animal feedreal-time polymerase chain reaction (PCR)-based method for the published
detection of food-borne pathogenshorizontal method for the detection of Shiga toxin-producing
Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups
Microbiology of food and animal feeding stuffshorizontal method for detection of hepatitis A virus published
and norovirus in food using real-time RT-PCRPart 1: method for quantitative determination
Microbiology of food and animal feeding stuffshorizontal method for detection of hepatitis A virus published
and norovirus in food using real-time RT-PCRPart 1: method for qualitative determination
Microbiology of food, feeding stuffs and environmental samplespolymerase chain reaction (PCR) for published
the detection of food-borne pathogensdetection of botulinum type A, B, E and F neurotoxin
producing clostridia
General requirements relating to molecular methods for detection and quantification of in discussion
micro-organismsdraft standard of PCR detection of enteropathogenic Yersinia spp.
Microbiology of food and animal feeding stuffsdetection of Vibrio parahaemolyticus in seafoods: Part in discussion
1quantitative determination of total, thermostable direct haemolysin (TDH) and thermostable
direct-related haemolysin (TRH) positive Vibrio parahaemolyticus using nucleic acid hybridisation

et al. 2003) and the working temperature of the lab may
influence the outcome of the experiment (Anonymous 2010).
It is necessary that entire analytical methods are validated.
In many cases, the validation study was focused on one step
only (e.g. PCR conditions) and thus gave no information on
Table 2 Performance characteristics to be evaluated during validation of
qualitative and quantitative alternative methods by comparison studies
(A) and interlaboratory studies (B) in accordance with ISO standard
16140:2003 Microbiology of food and animal feeding stuffsprotocol
for the validation of alternative methods
Qualitative methods Quantitative methods
how the validated PCR protocol would work in routine anal-
ysis. Therefore, it is recommended to select a certain sample,
an appropriate sample processing method and the most suit-
able PCR reagents and amplification parameters and then
perform validation of the whole process instead of individual
process steps (Hoorfar et al. 2004b).
It is necessary that the analytical methods are validated with
real-world samples. Many validation studies do not provide
sufficient information on the efficacy of the method because
only spiked samples are used, although there is growing concern
on the comparability of results obtained by spike experiments

A Table 3 Areas of uniformity in the protocols used for comparing Micro-

Relative accuracy
Relative specificity
Relative sensitivity
Relative detection level
Inclusivity and exclusivity
(selectivity)
Linearity
B
Relative accuracy
Percentage specificity
Percentage sensitivity
Accordance
Concordance
Concordance odds ratio
Relative accuracy
Specifity
Relative sensitivity
Detection and quantification limits
Inclusivity and exclusivity (selectivity)
Relative accuracy
Repeatibility (repeatability limit)
Reproducibility (reproducibility limit)
biological methods in the different validation methods according to
MoniQA European project working group coordinated by Paulin S
(Anonymous 2010)
Reference method (no particular method suggested by any validation
organisation for each pathogen)
Relevant food categories for each target pathogen (AOAC has
highlighted more categories than ISO or NordVal)
Number of required inocula levels (this ranges from 3 to 5 depending on
the organisation)
Number of strains required for sensitivity experiments (this ranges from
1 to 2 depending on the organisation)
Number of test portions
Performance indicators
Number of laboratories required to participate in interlaboratory and
collaborative trials
266
(healthy cultures) and those obtained in routine analysis
(stressed cultures) (Hoorfar et al. 2004b; Havelaar et al. 2010).
A lack of information also exists when it comes to quanti-
tative real-time PCR and reverse transcription real-time PCR.
Many publications do not provide sufficient detail on the
experimental conditions, and hence, reliable and unequivocal
interpretation is hardly possible. A set of guidelines describing
the Minimum Information for Publication of Quantitative
Real-Time PCR Experiments (MIQE) aims to overcome this
shortage by an increased consensus on the performance and
interpretation of real-time PCR protocols (Bustin et al. 2009).
For standardisation purposes, four areas (study design, tech-
nical detail, analysis methods and statistics) have to be defined
for any real-time PCR method. The MIQE guidelines consider
all of them, asking for details on the experimental design,
sample (processing, storage), nucleic acid extraction, reverse
transcription, the real-time PCR step and data analysis (Bustin
2010; Bustin et al. 2009).
A specific problem regards validation of reverse transcrip-
tion real-time PCR. The method is currently the gold standard
for the detection of (small) amounts of mRNA due to the
methods unchallenged simplicity, cost efficiency, accuracy
and availability (Pfaffl et al. 2002; Pugniere et al. 2011). As
this method combines reverse transcription with subsequent
real-time PCR, variations in results stem not only from real-
time PCR but also from RT yields. RT yield is influenced by
total RNA concentration, target template quantity and back-
ground DNA, isolation method and inhibitory substances like
myoglobin, collagen or lipids (Pugniere et al. 2011).
Once a standardised PCR/real-time PCR protocol is ready,
it cannot be considered as final but has to be continuously
revalidated in order to assess its sensitivity against newly
arising strains or to consider enhanced detection methods
(Malorny et al. 2003).
A new initiative was started in 2011 by AOAC, the
International Stakeholder Panel for Alternative Methods
(ISPAM). The purpose of this initiative is to develop
harmonised, internationally accepted standard validation guide-
lines for alternative (rapid) chemical and microbiological
methods, by leveraging global networks of experts to reach
consensus on an analytical validation protocol. Under this
initiative, AOAC has called together representatives from other
organisations that operate in the same fields, including ISO,
CEN, NordVal (Nordic Committee on Food Analysis, Oslo,
Norway), MicroVal (MicroVal, Delft, the Netherlands) and
AFNOR (French Standardization Organization, Paris, France).
In addition to the organisations, the panels also include repre-
sentatives from food and feed industry and kit and equipment
manufacturers as well as governments. All these stakeholders
use this type of methodology or have to base risk assessment on
the results obtained by using these methods on their samples.
One of the major goals of the panel is to come to a
harmonised approach on how to reduce the validation effort
Food Anal. Methods (2015) 8:255271
(e.g. fewer laboratories required or fewer samples required)
without significantly sacrificing the quality of the study,
allowing on one hand validation to take place faster, before
the methods are already obsolete, and, on the other hand, to
have mutual recognition of methods validated under the new
scheme, i.e. all organisations will accept the successful vali-
dation study without the need for repeating it for their organi-
sations approval.
ISPAM formed two working groups: a microbiology group
and a qualitative chemistry working group. The ISPAM micro-
biology group identified five high-priority areas for their future
work: reference methods, selection of food/category (sample
matrix), number of levels/samples/fractional positive, results
analysis and criteria/statistical analysis, number of samples/
replicates/method comparison and collaborative phase.
Cost of Molecular Methods for Food Analysis
Molecular methods for food analysis are currently mostly
available as kits, but the situation will probably change when
the necessary international norms are published. Laboratories
can then make use of the fact that molecular biological equip-
ment and biochemicals have become much more affordable in
recent years.
Currently, the use of kits or automated systems may seem
expensive, with prices as high as $10$15/7 EUR10 EUR
per test, sometimes over $25/18 EUR per test. The cost of
reagents and instruments used in rapid assays is very high,
$30,000$50,000/20,000 EUR35,000 EUR. Such costs are
affordable only for large companies. When it comes to detect-
ing micro-organisms by alternative or innovative rapid
methods, capital costs for automated PCR systems are rela-
tively high compared to those for some other rapid methods.
Consumable costs are also higher in comparison to rapid
culture-based techniques. However, there are considerable
cost benefits in terms of reduced technician labour time and
savings on training. There is a clear cost benefit in rapid test
results allowing faster HACCP verification and positive re-
lease of finished food products. A recent study carried out at
the German Federal Institute for Risk Assessment (BfR) con-
cluded that, taking all costs into account, real-time PCR could
be significantly less expensive than a conventional ISO cul-
ture method (http://www.rapidmicrobiology.com/test-method/
pcr-for-food-microbiology/).
Conclusion
In order to achieve full integration of molecular methods in
food safety control, certain drawbacks of PCR-methods still
need to be addressed and clarified, particularly the access to
the target cell by advanced sample treatment concepts and
Food Anal. Methods (2015) 8:255271
improved strategies to distinguish DNA of viable cells from
both DNA of dead or non-cultivable cells. In Europe, molec-
ular methods, either immunological or nucleic acid-based, are
currently used for preliminary and rapid screening of the
presence of pathogenic micro-organisms in food. The positive
results are considered presumptive and require further confir-
mation by traditional culture-based methods (Hoorfar 2011).
Also in USA, AOAC-approved rapid methods are used as
screening tools. Using first-line rapid testing and second-line
culture-dependent confirmations makes food testing costly,
consuming time and labour (Mandal et al. 2011). It has fur-
thermore led to the fact that molecular tests are used more and
more by FBOs but not by public health authorities. It would be
a clear step forward to a more efficient food safety system if
response actions could be accelerated up by accepting PCR-
based tests as reliable and definitive.
The slow implementation of novel technologies in food
microbiology is even more remarkable since a lot of compar-
isons between molecular and traditional cultural methods
demonstrated the equivalence of the data obtained from both
approaches to date (Gianfranceschi et al. 2014; Lofstrom and
Hoorfar 2012; Schultz et al. 2010; Zhang et al. 2011; Josefsen
et al. 2004; Abdulmawjood et al. 2003; Lubeck et al. 2003a;
Lubeck et al. 2003b; Josefsen et al. 2003; Fenicia et al. 2011;
Delibato et al. 2014). In order to support the implementation
of molecular methods, their standardisation through the re-
sponsible standardisation bodies should be prioritised, which
would facilitate the acceptance of nucleic acid-based food
analytics. It must by the ultimate goal that, when using mo-
lecular methods, the need to confirm positive results should be
limited to cases where a physical isolate is needed for epide-
miological or research purposes; if the physical isolate is not
required for subsequent studies, the positive result obtained by
molecular method should not require confirmation anymore.
Acknowledgments This research was supported by the framework of
the EU project funded by the 7th Framework Programme of the European
Union Safe Food for EuropeCoordination of research activities and
Dissemination of research results of EC funded research on food safety
(project acronym: FOODSEG) Grant agreement no. 266061. This publi-
cation reflects the views only of the authors, and the European Commis-
sion cannot be held responsible for any use which may be made of the
information contained therein.
Conflict of Interest No financial relationship with other institutions or
private industry has influenced the results of this study. D. De Medici has
no conflict of interest, T. Kuchta has no conflict of interest, R. Knutsson
has no conflict of interest, A. Angelov has no conflict of interest, B.
Auricchio has no conflict of interest, M. Barbanera has no conflict of
interest, C. Diaz-Amigo has no conflict of interest, A. Fiore has no
conflict of interest, E. Kudirkiene has no conflict of interest, A. Hohl
has no conflict of interest, D. Horvatek Tomic has no conflict of interest,
V. Gotcheva has no conflict of interest, B. Popping has no conflict of
interest, E. Prukner-Radovcic has no conflict of interest, S. Scaramaglia
has no conflict of interest, P. Siekel has no conflict of interest, K.A. To has
no conflict of interest and M. Wagner has no conflict of interest. This
article does not contain any studies with human or animal subjects.
267
References
Abdulmawjood A, Bulte M, Cook N, Roth S, Schonenbrucher H, Hoorfar
J (2003) Toward an international standard for PCR-based detection
of Escherichia coli O157. Part 1. Assay development and multi-
center validation. J Microbiol Methods 55(3):775786
Abdulmawjood A, Bulte M, Roth S, Schonenbrucher H, Cook N,
DAgostino M, Burkhard M, Jordan K, Pelkonen S, Hoorfar J
(2004) Toward an international standard for PCR-based detection of
foodborne Escherichia coli O157: validation of the PCR-based meth-
od in a multicenter interlaboratory trial. J AOAC Int 87(4):856860
Amagliani G, Petruzzelli A, Omiccioli E, Tonucci F, Magnani M, Brandi
G (2012) Microbiological surveillance of a bovine raw milk farm
through multiplex real-time PCR. Foodborne Pathog Dis 9(5):406
411. doi:10.1089/fpd.2011.1041
Anonymous (2010) Moniqa project- Executive summary 2010. http://
www.moniqa.eu/sites/moniqa.eu/files/pagefiles/exec_glossy_10_lr.
pdf
Aprodu I, Walcher G, Schelin J, Hein I, Norling B, Radstrom P, Nicolau
A, Wagner M (2011) Advanced sample preparation for the molec-
ular quantification of Staphylococcus aureus in artificially and nat-
urally contaminated milk. Int J Food Microbiol 145(Suppl 1):S61
S65. doi:10.1016/j.ijfoodmicro.2010.09.018
Bidawid S, Farber JM, Sattar SA (2000) Rapid concentration and detec-
tion of hepatitis A virus from lettuce and strawberries. J Virol
Methods 88(2):175185
Birch L, Dawson CE, Cornett JH, Keer JT (2001) A comparison of
nucleic acid amplification techniques for the assessment of bacterial
viability. Lett Appl Microbiol 33(4):296301
Brehm-Stecher B, Young C, Jaykus LA, Tortorello ML (2009) Sample
preparation: the forgotten beginning. J Food Prot 72(8):17741789
Brezna B, Hudecova L, Kuchta T (2006) Detection of pea in food by real-
time polymerase chain reaction (PCR). Eur Food Res Technol 222:
600603
Bustin SA (2010) Why the need for qPCR publication guidelines?the
case for MIQE. Methods 50(4):217226. doi:10.1016/j.ymeth.
2009.12.006
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M,
Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J,
Wittwer CT (2009) The MIQE guidelines: minimum information
for publication of quantitative real-time PCR experiments. Clin
Chem 55(4):611622. doi:10.1373/clinchem.2008.112797
Butot S, Putallaz T, Croquet C, Lamothe G, Meyer R, Joosten H, Sanchez
G (2007a) Attachment of enteric viruses to bottles. Appl Environ
Microbiol 73(16):51045110. doi:10.1128/AEM.00450-07
Butot S, Putallaz T, Sanchez G (2007b) Procedure for rapid concentration
and detection of enteric viruses from berries and vegetables. Appl
Environ Microbiol 73(1):186192. doi:10.1128/AEM.01248-06
Butot S, Patallaz T, Sanchez G (2013) Improvement of procedure for
HAV detection in bottled water. Food Anal Methods 6:270273
Chancellor DD, Tyagi S, Bazaco MC, Bacvinskas S, Chancellor MB,
Dato VM, de Miguel F (2006) Green onions: potential mechanism
for hepatitis A contamination. J Food Prot 69(6):14681472
CodexAlimentarius (2012) Proposed draft revision of the principles for
the establishment and application of microbiological criteria for
foods. FAO/OMS, Rome
Costafreda MI, Bosch A, Pinto RM (2006) Development, evaluation, and
standardization of a real-time TaqMan reverse transcription-PCR
assay for quantification of hepatitis A virus in clinical and shellfish
samples. Appl Environ Microbiol 72(6):38463855. doi:10.1128/
AEM.02660-05
Coudray-Meunier C, Fraisse A, Martin-Latil S, Guillier L, Perelle S
(2013) Discrimination of infectious hepatitis A virus and rotavirus
by combining dyes and surfactants with RT-qPCR. BMC Microbiol
13:216. doi:10.1186/1471-2180-13-216
268
Coutard F, Pommepuy M, Loaec S, Hervio-Heath D (2005) mRNA
detection by reverse transcription-PCR for monitoring viability
and potential virulence in a pathogenic strain of Vibrio
parahaemolyticus in viable but nonculturable state. J Appl
Microbiol 98(4):951961. doi:10.1111/j.1365-2672.2005.02534.x
Croci L, Dubois E, Cook N, De Medici D, Schultz AC, China B, Rutjes
SA, Hoorfar H, Van der Poel WH (2008) Current methods for
extraction and concentration of enteric viruses from fresh fruit and
vegetables: towards international standards. Food Anal Methods 1:
7384
DAgostino M, Wagner M, Vazquez-Boland JA, Kuchta T, Karpiskova
R, Hoorfar J, Novella S, Scortti M, Ellison J, Murray A, Fernandes I,
Kuhn M, Pazlarova J, Heuvelink A, Cook N (2004) A validated
PCR-based method to detect Listeria monocytogenes using raw milk
as a food modeltowards an international standard. J Food Prot
67(8):16461655
DUrso OF, Poltronieri P, Marsigliante S, Storelli C, Hernandez M,
Rodriguez-Lazaro D (2009) A filtration-based real-time PCR meth-
od for the quantitative detection of viable Salmonella enterica and
Listeria monocytogenes in food samples. Food Microbiol 26(3):
311316. doi:10.1016/j.fm.2008.12.006
De Medici D, Croci L, Di Pasquale S, Fiore A, Toti L (2001) Detecting
the presence of infectious hepatitis A virus in molluscs positive to
RT-nested-PCR. Lett Appl Microbiol 33(5):362366
De Medici D, Anniballi F, Wyatt GM, Lindstrom M, Messelhausser U,
Aldus CF, Delibato E, Korkeala H, Peck MW, Fenicia L (2009)
Multiplex PCR for detection of botulinum neurotoxin-producing
clostridia in clinical, food, and environmental samples. Appl
Environ Microbiol 75(20):64576461. doi:10.1128/AEM.00805-09
Delibato E, Rodriguez Lazaro D, Gianfranceschi M, De Cesare A, Comin
D, Gattuso A, Hernandez M, Sonnessa M, Pasquali F, Sreter-Lancz
Z, Saiz-Abajo MJ, Perez-De-Juan J, Butron J, Prukner-Radovcic E,
Horvatek Tomic D, Johannessen GS, Jakociune D, Olsen JE,
Chemaly M, Le Gall F, Gonzalez-Garcia P, Lettini AA, Lukac M,
Quesne S, Zampieron C, De Santis P, Lovari S, Bertasi B, Pavoni E,
Proroga YT, Capuano F, Manfreda G, De Medici D (2014)
European validation of real-time PCR method for detection of
Salmonella spp. in pork meat. Int J Food Microbiol. doi:10.1016/j.
ijfoodmicro.2014.01.005
Di Pasquale S, Paniconi M, De Medici D, Suffredini E, Croci L (2009)
Duplex real time PCR for the detection of hepatitis A virus in
shellfish using Feline Calicivirus as a process control. J Virol
Methods 163(1):96100. doi:10.1016/j.jviromet.2009.09.003
Di Pasquale S, Paniconi M, Auricchio B, Orefice L, Schultz AC, De
Medici D (2010) Comparison of different concentration methods for
the detection of hepatitis A virus and calicivirus from bottled natural
mineral waters. J Virol Methods 165(1):5763. doi:10.1016/j.
jviromet.2010.01.003
Duizer E, Schwab KJ, Neill FH, Atmar RL, Koopmans MP, Estes MK
(2004) Laboratory efforts to cultivate noroviruses. J Gen Virol 85(Pt
1):7987
EFSA (2011) Scientific opinion on an update on the present knowledge
on the occurrence and control of foodborne viruses. EFSA J 9(7):
2190
El-Senousy WM, Costafreda MI, Pinto RM, Bosch A (2013) Method
validation for norovirus detection in naturally contaminated irriga-
tion water and fresh produce. Int J Food Microbiol 167(1):7479.
doi:10.1016/j.ijfoodmicro.2013.06.023
Fach P, Micheau P, Mazuet C, Perelle S, Popoff M (2009) Development
of real-time PCR tests for detecting botulinum neurotoxins A, B, E,
F producing Clostridium botulinum, Clostridium baratii and
Clostridium butyricum. J Appl Microbiol 107(2):465473. doi:10.
1111/j.1365-2672.2009.04215.x
Fenicia L, Fach P, van Rotterdam BJ, Anniballi F, Segerman B, Auricchio
B, Delibato E, Hamidjaja RA, Wielinga PR, Woudstra C, Agren J,
De Medici D, Knutsson R (2011) Towards an international standard
Food Anal. Methods (2015) 8:255271
for detection and typing botulinum neurotoxin-producing Clostridia
types A, B, E and F in food, feed and environmental samples: a
European ring trial study to evaluate a real-time PCR assay. Int J
Food Microbiol 145(Suppl 1):S152S157. doi:10.1016/j.
ijfoodmicro.2011.02.001
Flekna G, Stefanic P, Wagner M, Smulders FJ, Mozina SS, Hein I (2007)
Insufficient differentiation of live and dead Campylobacter jejuni
and Listeria monocytogenes cells by ethidium monoazide (EMA)
compromises EMA/real-time PCR. Res Microbiol 158(5):405412.
doi:10.1016/j.resmic.2007.02.008
Fricker M, Messelhausser U, Busch U, Scherer S, Ehling-Schulz M (2007)
Diagnostic real-time PCR assays for the detection of emetic Bacillus
cereus strains in foods and recent food-borne outbreaks. Appl
Environ Microbiol 73(6):18921898. doi:10.1128/AEM.02219-06
Fukushima H, Katsube K, Hata Y, Kishi R, Fujiwara S (2007) Rapid
separation and concentration of food-borne pathogens in food sam-
ples prior to quantification by viable-cell counting and real-time
PCR. Appl Environ Microbiol 73(1):92100. doi:10.1128/AEM.
01772-06
Gianfranceschi MV, Rodriguez-Lazaro D, Hernandez M, Gonzalez-
Garcia P, Comin D, Gattuso A, Delibato E, Sonnessa M, Pasquali
F, Prencipe V, Sreter-Lancz Z, Saiz-Abajo MJ, Perez-De-Juan J,
Butron J, Kozacinski L, Tomic DH, Zdolec N, Johannessen GS,
Jakociune D, Olsen JE, De Santis P, Lovari S, Bertasi B, Pavoni E,
Paiusco A, De Cesare A, Manfreda G, De Medici D (2014)
European validation of a real-time PCR-based method for detection
of Listeria monocytogenes in soft cheese. Int J Food Microbiol. doi:
10.1016/j.ijfoodmicro.2013.12.021
Gonzalez-Escalona N, Hammack TS, Russell M, Jacobson AP, De Jesus
AJ, Brown EW, Lampel KA (2009) Detection of live Salmonella sp.
cells in produce by a TaqMan-based quantitative reverse transcrip-
tase real-time PCR targeting invA mRNA. Appl Environ Microbiol
75(11):37143720. doi:10.1128/AEM.02686-08
Graiver DA, Saunders SE, Topliff CL, Kelling CL, Bartelt-Hunt SL
(2010) Ethidium monoazide does not inhibit RT-PCR amplification
of nonviable avian influenza RNA. J Virol Methods 164(12):51
54. doi:10.1016/j.jviromet.2009.11.024
Greisen K, Loeffelholz M, Purohit A, Leong D (1994) PCR primers and
probes for the 16S rRNA gene of most species of pathogenic
bacteria, including bacteria found in cerebrospinal fluid. J Clin
Microbiol 32(2):335351
Havelaar AH, Brul S, de Jong A, de Jonge R, Zwietering MH, Ter Kuile
BH (2010) Future challenges to microbial food safety. Int J Food
Microbiol 139(Suppl 1):S79S94. doi:10.1016/j.ijfoodmicro.2009.
10.015
Hellyer TJ, DesJardin LE, Teixeira L, Perkins MD, Cave MD, Eisenach
KD (1999) Detection of viable Mycobacterium tuberculosis by
reverse transcriptase-strand displacement amplification of mRNA.
J Clin Microbiol 37(3):518523
Hofstra H, van der Vossen JM, van der Plas J (1994) Microbes in food
processing technology. FEMS Microbiol Rev 15(23):175183
Hoorfar J (2011) Rapid detection, characterization, and enumeration of
foodborne pathogens. APMIS Suppl 133:124. doi:10.1111/j.1600-
0463.2011.02767.x
Hoorfar J, Malorny B, Abdulmawjood A, Cook N, Wagner M, Fach P
(2004a) Practical considerations in design of internal amplification
controls for diagnostic PCR assays. J Clin Microbiol 42(5):1863
1868
Hoorfar J, Wolffs P, Radstrom P (2004b) Diagnostic PCR: validation and
sample preparation are two sides of the same coin. APMIS 112(11
12):808814
Huang P, Farkas T, Zhong W, Tan M, Thornton S, Morrow AL, Jiang X
(2005) Norovirus and histo-blood group antigens: demonstration of
a wide spectrum of strain specificities and classification of two
major binding groups among multiple binding patterns. J Virol
79(11):67146722. doi:10.1128/JVI.79.11.6714-6722.2005
Food Anal. Methods (2015) 8:255271
Hyeon JY, Chon JW, Park C, Lee JB, Choi IS, Kim MS, Seo KH (2011)
Rapid detection method for hepatitis A virus from lettuce by a
combination of filtration and integrated cell culture-real-time reverse
transcription PCR. J Food Prot 74(10):17561761. doi:10.4315/
0362-028X.JFP-11-155
ISO/IEC (2004) ISO/IEC Guide 2:2004, Standardization and related
activitiesgeneral vocabulary
Jakociune D, Pasquali F, da Silva CS, Lofstrom C, Hoorfar J, Klein G,
Manfreda G, Olsen JE (2013) Enumeration of salmonellae in table
eggs, pasteurized egg products, and egg-containing dishes by using
quantitative real-time PCR. Appl Environ Microbiol 80(5):1616
1622. doi:10.1128/AEM.03360-13
Josefsen MH, Lambertz ST, Jensen S, Hoorfar J (2003) Food-PCR.
Validation and standardization of diagnostic PCR for detection of
Yersinia enterocolitica and other foodborne pathogens. Adv Exp
Med Biol 529:443449. doi:10.1007/0-306-48416-1_88
Josefsen MH, Cook N, DAgostino M, Hansen F, Wagner M, Demnerova
K, Heuvelink AE, Tassios PT, Lindmark H, Kmet V, Barbanera M,
Fach P, Loncarevic S, Hoorfar J (2004) Validation of a PCR-based
method for detection of food-borne thermotolerant campylobacters
in a multicenter collaborative trial. Appl Environ Microbiol 70(7):
43794383. doi:10.1128/AEM.70.7.4379-4383.2004
Josefsen MH, Lofstrom C, Hansen TB, Christensen LS, Olsen JE,
Hoorfar J (2010) Rapid quantification of viable Campylobacter
bacteria on chicken carcasses, using real-time PCR and
propidium monoazide treatment, as a tool for quantitative risk
assessment. Appl Environ Microbiol 76(15):50975104. doi:10.
1128/AEM.00411-10
Jothikumar N, Lowther JA, Henshilwood K, Lees DN, Hill VR, Vinje J
(2005) Rapid and sensitive detection of noroviruses by using
TaqMan-based one-step reverse transcription-PCR assays and ap-
plication to naturally contaminated shellfish samples. Appl Environ
Microbiol 71(4):18701875. doi:10.1128/AEM.71.4.1870-1875.
2005
Kim SY, Ko G (2012) Using propidium monoazide to distinguish be-
tween viable and nonviable bacteria, MS2 and murine norovirus.
Lett Appl Microbiol 55(3):182188. doi:10.1111/j.1472-765X.
2012.03276.x
Kim K, Katayama H, Kitajima M, Tohya Y, Ohgaki S (2011)
Development of a real-time RT-PCR assay combined with ethidium
monoazide treatment for RNA viruses and its application to detect
viral RNA after heat exposure. Water Sci Technol 63(3):502507.
doi:10.2166/wst.2011.249
Knight A, Li D, Uyttendaele M, Jaykus LA (2012) A critical review of
methods for detecting human noroviruses and predicting their infec-
tivity. Crit Rev Microbiol. doi:10.3109/1040841X.2012.709820
Knutsson R, van Rotterdam B, Fach P, De Medici D, Fricker M, Lofstrom
C, Agren J, Segerman B, Andersson G, Wielinga P, Fenicia L, Skiby
J, Schultz AC, Ehling-Schulz M (2011) Accidental and deliberate
microbiological contamination in the feed and food chainshow
biotraceability may improve the response to bioterrorism. Int J Food
Microbiol 145(Suppl 1):S123S128. doi:10.1016/j.ijfoodmicro.
2010.10.011
Kobayashi S, Natori K, Takeda N, Sakae K (2004) Immunomagnetic
capture rt-PCR for detection of norovirus from foods implicated in a
foodborne outbreak. Microbiol Immunol 48(3):201204
Kobayashi H, Oethinger M, Tuohy MJ, Procop GW, Hall GS, Bauer TW
(2009) Limiting false-positive polymerase chain reaction results:
detection of DNA and mRNA to differentiate viable from dead
bacteria. Diagn Microbiol Infect Dis 64(4):445447. doi:10.1016/j.
diagmicrobio.2009.04.004
Konduru K, Kaplan GG (2006) Stable growth of wild-type hepatitis A
virus in cell culture. J Virol 80(3):13521360. doi:10.1128/JVI.80.3.
1352-1360.2006
Koopmans M, von Bonsdorff CH, Vinje J, de Medici D, Monroe S (2002)
Foodborne viruses. FEMS Microbiol Rev 26(2):187205
269
Kornacki JL, Johnson JL (2001) Enterobacteriacae, coliform and
Escherichia coli as quality and safety indicators. Chapter 8. In:
APHA (ed) Compendium of methods for the microbiological ex-
amination, 4th edn. APHA, Washington D.C
Kramer N, Lofstrom C, Vigre H, Hoorfar J, Bunge C, Malorny B (2011)
A novel strategy to obtain quantitative data for modelling: combined
enrichment and real-time PCR for enumeration of salmonellae from
pig carcasses. Int J Food Microbiol 145(Suppl 1):S86S95. doi:10.
1016/j.ijfoodmicro.2010.08.026
Krascsenicsova K, Kaclikova E, Kuchta T (2006) Growth of Salmonella
enterica in model mixed cultures during a two-step enrichment. New
Microbiol 29(4):261267
Kuchta T, Knutsson R, Fiore A, Kudirkiene E, Hhl A, Horvatek Tomic
D, Gotcheva V, Ppping B, Scaramaglia S, To KA, Wagner M, De
Medici D (2014) A decade with nucleic acid-based microbiological
methods in safety control of foods. Lett Appl Microbiol. doi:10.
1111/lam.12283
Lau HK, Clotilde LM, Lin AP, Hartman GL, Lauzon CR (2012)
Comparison of IMS platforms for detecting and recovering
Escherichia coli O157 and Shigella flexneri in foods. J Lab
Autom. doi:10.1177/2211068212468583
Le Guyader F, Loisy F, Atmar RL, Hutson AM, Estes MK, Ruvoen-
Clouet N, Pommepuy M, Le Pendu J (2006) Norwalk virus-specific
binding to oyster digestive tissues. Emerg Infect Dis 12(6):931936
Lees D (2010) International standardisation of a method for detection of
human pathogenic viruses in molluscan shellfish. Food Anal
Method 2010:146155
Lindstrom M, Keto R, Markkula A, Nevas M, Hielm S, Korkeala H
(2001) Multiplex PCR assay for detection and identification of
Clostridium botulinum types A, B, E, and F in food and fecal
material. Appl Environ Microbiol 67(12):56945699. doi:10.1128/
AEM.67.12.5694-5699.2001
Liu Y, Wang C, Fung C, Li XF (2010) Quantification of viable but
nonculturable Escherichia coli O157:H7 by targeting the rpoS
mRNA. Anal Chem 82(7):26122615. doi:10.1021/ac1003272
Lofstrom C, Hoorfar J (2012) Validation of an open-formula, diagnostic
real-time PCR method for 20-h detection of Salmonella in animal
feeds. Vet Microbiol 158(34):431435. doi:10.1016/j.vetmic.2012.
02.026
Lombard B, Leclercq A (2009) The role of standardization bodies in the
harmonization of analytical methods in food microbiology. In:
Barbosa-Canovas G, Mortimer A, Lineback D, Spiess W, Buckle
K, Colonna P (eds) Global issues in food science and technology.
Elsevier Academic Press, Burlington, pp 177197
Lubeck PS, Cook N, Wagner M, Fach P, Hoorfar J (2003a) Toward an
international standard for PCR-based detection of food-borne
thermotolerant Campylobacters: validation in a multicenter collab-
orative trial. Appl Environ Microbiol 69(9):56705672
Lubeck PS, Wolffs P, On SL, Ahrens P, Radstrom P, Hoorfar J (2003b)
Toward an international standard for PCR-based detection of food-
borne thermotolerant campylobacters: assay development and ana-
lytical validation. Appl Environ Microbiol 69(9):56645669
Malorny B, Tassios PT, Radstrom P, Cook N, Wagner M, Hoorfar J
(2003) Standardization of diagnostic PCR for the detection of
foodborne pathogens. Int J Food Microbiol 83(1):3948
Malorny B, Cook N, DAgostino M, De Medici D, Croci L,
Abdulmawjood A, Fach P, Karpiskova R, Aymerich T, Kwaitek K,
Hoorfar J (2004) Multicenter validation of PCR-based method for
detection of Salmonella in chicken and pig samples. J AOAC Int
87(4):861866
Mancusi R, Trevisani M (2014) Enumeration of verocytotoxigenic
Escherichia coli (VTEC) O157 and O26 in milk by quantitative
PCR. Int J Food Microbiol. doi:10.1016/j.ijfoodmicro.2014.03.020
Mandal PK, Biswas AK, Choi K, Pal UK (2011) Method for rapid
detection of foodborne pathogens: an overview. Am J Food
Technol 6:87102
270
Manfreda G, De Cesare A (2014) The challenge of defining risk-based
metrics to improve food safety: inputs from the BASELINE project.
Int J Food Microbiol. doi:10.1016/j.ijfoodmicro.2014.01.013
Mayrl E, Roeder B, Mester P, Wagner M, Rossmanith P (2009) Broad
range evaluation of the matrix solubilization (matrix lysis) strategy
for direct enumeration of foodborne pathogens by nucleic acids
technologies. J Food Prot 72(6):12251233
Mester P, Wagner M, Rossmanith P (2010a) Biased spectroscopic protein
quantification in the presence of ionic liquids. Anal Bioanal Chem
397(5):17631766. doi:10.1007/s00216-010-3755-z
Mester P, Wagner M, Rossmanith P (2010b) Use of ionic liquid-based
extraction for recovery of Salmonella Typhimurium and Listeria
monocytogenes from food matrices. J Food Prot 73(4):680687
Minami J, Yoshida K, Soejima T, Yaeshima T, Iwatsuki K (2010) New
approach to use ethidium bromide monoazide as an analytical tool. J
Appl Microbiol 109(3):900909. doi:10.1111/j.1365-2672.2010.
04716.x
Minami J, Soejima T, Yaeshima T, Iwatsuki K (2012) Direct real-time
PCR with ethidium monoazide: a method for the rapid detection of
viable Cronobacter sakazakii in powdered infant formula. J Food
Prot 75(9):15721579. doi:10.4315/0362-028X.JFP-12-015
Morton V, Jean J, Farber J, Mattison K (2009) Detection of noroviruses in
ready-to-eat foods by using carbohydrate-coated magnetic beads.
Appl Environ Microbiol 75(13):46414643. doi:10.1128/AEM.
00202-09
Nocker A, Camper AK (2009) Novel approaches toward preferential
detection of viable cells using nucleic acid amplification techniques.
FEMS Microbiol Lett 291(2):137142. doi:10.1111/j.1574-6968.
2008.01429.x
Nocker A, Cheung CY, Camper AK (2006) Comparison of propidium
monoazide with ethidium monoazide for differentiation of live vs.
dead bacteria by selective removal of DNA from dead cells. J
Microbiol Methods 67(2):310320. doi:10.1016/j.mimet.2006.04.
015
Nocker A, Sossa-Fernandez P, Burr MD, Camper AK (2007) Use of
propidium monoazide for live/dead distinction in microbial ecology.
Appl Environ Microbiol 73(16):51115117. doi:10.1128/AEM.
02987-06
Nogva HK, Dromtorp SM, Nissen H, Rudi K (2003) Ethidium
monoazide for DNA-based differentiation of viable and dead bacte-
ria by 5-nuclease PCR. Biotechniques 34(4):804808, 810, 812-
803
Nuanualsuwan S, Cliver DO (2002) Pretreatment to avoid positive RT-
PCR results with inactivated viruses. J Virol Methods 104(2):217
225
Nuanualsuwan S, Cliver DO (2003) Capsid functions of inactivated
human picornaviruses and feline calicivirus. Appl Environ
Microbiol 69(1):350357
Nuanualsuwan S, Mariam T, Himathongkham S, Cliver DO (2002)
Ultraviolet inactivation of feline calicivirus, human enteric viruses
and coliphages. Photochem Photobiol 76(4):406410
Ogorzaly L, Bertrand I, Paris M, Maul A, Gantzer C (2010) Occurrence,
survival, and persistence of human adenoviruses and F-specific
RNA phages in raw groundwater. Appl Environ Microbiol 76(24):
80198025. doi:10.1128/AEM.00917-10
Ogorzaly L, Bonot S, Moualij BE, Zorzi W, Cauchie HM (2013)
Development of a quantitative immunocapture real-time PCR assay
for detecting structurally intact adenoviral particles in water. J Virol
Methods 194(12):235241. doi:10.1016/j.jviromet.2013.07.009
Olsvik O, Popovic T, Skjerve E, Cudjoe KS, Hornes E, Ugelstad J, Uhlen
M (1994) Magnetic separation techniques in diagnostic microbiol-
ogy. Clin Microbiol Rev 7(1):4354
Papafragkou E, Plante M, Mattison K, Bidawid S, Karthikeyan K, Farber
JM, Jaykus LA (2008) Rapid and sensitive detection of hepatitis A
virus in representative food matrices. J Virol Methods 147(1):177
187. doi:10.1016/j.jviromet.2007.08.024
Food Anal. Methods (2015) 8:255271
Parshionikar S, Laseke I, Fout GS (2010) Use of propidium monoazide in
reverse transcriptase PCR to distinguish between infectious and
noninfectious enteric viruses in water samples. Appl Environ
Microbiol 76(13):43184326. doi:10.1128/AEM.02800-09
Pavoni E, Consoli M, Suffredini E, Arcangeli G, Serracca L, Battistini R,
Rossini I, Croci L, Losio MN (2013) Noroviruses in seafood: a 9-
year monitoring in Italy. Foodborne Pathog Dis 10(6):533539. doi:
10.1089/fpd.2012.1399
Perelle S, Cavellini L, Burger C, Blaise-Boisseau S, Hennechart-Collette
C, Merle G, Fach P (2009) Use of a robotic RNA purification
protocol based on the NucliSens easyMAG for real-time RT-PCR
detection of hepatitis A virus in bottled water. J Virol Methods
157(1):8083. doi:10.1016/j.jviromet.2008.11.022
Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software
tool (REST) for group-wise comparison and statistical analysis of
relative expression results in real-time PCR. Nucleic Acids Res
30(9):e36
Postollec F, Falentin H, Pavan S, Combrisson J, Sohier D (2011) Recent
advances in quantitative PCR (qPCR) applications in food microbi-
ology. Food Microbiol 28(5):848861. doi:10.1016/j.fm.2011.02.008
Pugniere P, Banzet S, Chaillou T, Mouret C, Peinnequin A (2011) Pitfalls
of reverse transcription quantitative polymerase chain reaction stan-
dardization: volume-related inhibitors of reverse transcription. Anal
Biochem 415(2):151157. doi:10.1016/j.ab.2011.04.008
Radstrom P, Knutsson R, Wolffs P, Lovenklev M, Lofstrom C (2004) Pre-
PCR processing: strategies to generate PCR-compatible samples.
Mol Biotechnol 26(2):133146. doi:10.1385/MB:26:2:133
Radstrom P, Lofstrom C, Lovenklev M, Knutsson R, Wolffs P (2008)
Strategies for overcoming PCR inhibition. CSH Protoc 2008:pdb
top20
Reichert-Schwillinsky F, Pin C, Dzieciol M, Wagner M, Hein I (2009)
Stress- and growth rate-related differences between plate count and
real-time PCR data during growth of Listeria monocytogenes. Appl
Environ Microbiol 75(7):21322138. doi:10.1128/AEM.01796-08
Reynolds KA (2004) Integrated cell culture/PCR for detection of enteric
viruses in environmental samples. Methods Mol Biol 268:6978.
doi:10.1385/1-59259-766-1:069
Richards GP (1999) Limitations of molecular biological techniques for
assessing the virological safety of foods. J Food Prot 62(6):691697
Rizzo C, Alfonsi V, Bruni R, Busani L, Ciccaglione A, De Medici D, Di
Pasquale S, Equestre M, Escher M, Montano-Remacha M, Scavia
G, Taffon S, Carraro V, Franchini S, Natter B, Augschiller M, Tosti
M (2014) Central Task Force on Hepatitis A. Ongoing outbreak of
hepatitis A in Italy: preliminary report as of 31 May 2013. Euro
Surveill 18(27). Available online: http://www.eurosurveillance.org/
ViewArticle.aspx?ArticleId=20518
Rodriguez-Lazaro D, Hernandez M (2013) Real-time PCR in food sci-
ence: introduction. Curr Issues Mol Biol 15(2):2538
Rodrguez-Lzaro D, Lombard B, Smith H, Rzezutka A, DAgostino M,
Helmuth R, Schroeter A, Malorny B, Miko A, Guerra B, Davison J,
Kobilinsky A, Hernndez M, Bertheau Y, Cook N (2007) Trends in
analytical methodology in food safety and quality: monitoring mi-
croorganisms and genetically modified organisms. Trends Food Sci
Technol 18:306319
Rodriguez-Lazaro D, Cook N, Hernandez M (2013) Real-time PCR in
food science: PCR diagnostics. Curr Issues Mol Biol 15(2):3944
Rodriguez-Lazaro D, Gonzalez-Garca P, Delibato E, De Medici D,
Garca-Gimeno RM, Valero A, Hernandez M (2014) Next day
Salmonella spp. detection method based on real-time PCR for meat,
dairy and vegetable food products. Int J Food Microbiol. doi:10.
1016/j.ijfoodmicro.2014.03.021
Rosenquist H, Bengtsson A, Hansen TB (2007) A collaborative study on
a Nordic standard protocol for detection and enumeration of
thermotolerant Campylobacter in food (NMKL 119, 3. Ed., 2007).
Int J Food Microbiol 118(2):201213. doi:10.1016/j.ijfoodmicro.
2007.07.037
Food Anal. Methods (2015) 8:255271
Rossmanith P, Wagner M (2010) Sample preparation for the detection of
foodborne pathogens by molecular biological methods. In: Brul S,
Fratamico PM, McMeekin TA (eds) Tracing pathogens in the food
chain. Woodhead, Cambridge
Rossmanith P, Wagner M (2011) The challenge to quantify Listeria
monocytogenesa model leading to new aspects in molecular bio-
logical food pathogen detection. J Appl Microbiol 110(3):605617.
doi:10.1111/j.1365-2672.2010.04915.x
Rossmanith P, Suss B, Wagner M, Hein I (2007) Development of matrix
lysis for concentration of gram positive bacteria from food and
blood. J Microbiol Methods 69(3):504511. doi:10.1016/j.mimet.
2007.03.003
Rossmanith P, Mester P, Fruhwirth K, Fuchs S, Wagner M (2011) Proof
of concept for recombinant cellular controls in quantitative molec-
ular pathogen detection. Appl Environ Microbiol 77(7):25312533.
doi:10.1128/AEM.02601-10
Rudi K, Moen B, Dromtorp SM, Holck AL (2005) Use of ethidium
monoazide and PCR in combination for quantification of viable
and dead cells in complex samples. Appl Environ Microbiol 71(2):
10181024. doi:10.1128/AEM.71.2.1018-1024.2005
Sadovski AY, Fattal B, Goldberg D, Katzenelson E, Shuval HI (1978)
High levels of microbial contamination of vegetables irrigated with
wastewater by the drip method. Appl Environ Microbiol 36(6):824
830
Sanchez G, Bosch A, Pinto RM (2007) Hepatitis A virus detection in
food: current and future prospects. Lett Appl Microbiol 45(1):15.
doi:10.1111/j.1472-765X.2007.02140.x
Sanchez G, Elizaquivel P, Aznar R (2013) Discrimination of infectious
hepatitis A viruses by propidium monoazide real-time RT-PCR.
Food Environ Virol 4(1):2125. doi:10.1007/s12560-011-9074-5
Scherer K, Mde D, Ellerbroek L, Schulenburg J, Johne R, Klein G
(2008) Application of a swab sampling method for the detection of
norovirus and rotavirus to artificially contaminated food and envi-
ronmental surfaces. Food Environ Virol 1:4249
Schoder D, Schmalwieser A, Schauberger G, Kuhn M, Hoorfar J, Wagner
M (2003) Physical characteristics of six new thermocyclers. Clin
Chem 49(6 Pt 1):960963
Schultz AC, Perelle S, Di Pasquale S, Kovac K, De Medici D, Fach P,
Sommer HM, Hoorfar J (2010) Collaborative validation of a rapid
method for efficient virus concentration in bottled water. Int J Food
Microbiol 145(Suppl 1):S158S166. doi:10.1016/j.ijfoodmicro.
2010.07.030
Schultz AC, Perelle S, Di Pasquale S, Kovac K, De Medici D, Fach P,
Sommer HM, Hoorfar J (2011) Collaborative validation of a rapid
method for efficient virus concentration in bottled water. Int J Food
Microbiol 145(Suppl 1):S158S166. doi:10.1016/j.ijfoodmicro.
2010.07.030
Shan XC, Wolffs P, Griffiths MW (2005) Rapid and quantitative detection
of hepatitis A virus from green onion and strawberry rinses by use of
real-time reverse transcription-PCR. Appl Environ Microbiol 71(9):
56245626. doi:10.1128/AEM.71.9.5624-5626.2005
Shields MJ, Hahn KR, Janzen TW, Goji N, Thomas MC, Kingombe CB,
Paquet C, Kell AJ, Amoako KK (2012) Immunomagnetic capture of
Bacillus anthracis spores from food. J Food Prot 75(7):12431248.
doi:10.4315/0362-028X.JFP-12-048
Soejima T, Schlitt-Dittrich F, Yoshida S (2011) Polymerase chain reaction
amplification length-dependent ethidium monoazide suppression
power for heat-killed cells of Enterobacteriaceae. Anal Biochem
418(1):3743. doi:10.1016/j.ab.2011.06.027
Stals A, Mathijs E, Baert L, Botteldoorn N, Denayer S, Mauroy A,
Scipioni A, Daube G, Dierick K, Herman L, Van Coillie E, Thiry
E, Uyttendaele M (2013) Molecular detection and genotyping of
noroviruses. Food Environ Virol 4(4):153167. doi:10.1007/
s12560-012-9092-y
271
Stevens KA, Jaykus LA (2004) Direct detection of bacterial pathogens in
representative dairy products using a combined bacterial
concentration-PCR approach. J Appl Microbiol 97(6):11151122.
doi:10.1111/j.1365-2672.2004.02393.x
Suffredini E, Lanni L, Arcangeli G, Pepe T, Mazzette R, Ciccaglioni G,
Croci L (2014) Qualitative and quantitative assessment of viral
contamination in bivalve molluscs harvested in Italy. Int J Food
Microbiol. doi:10.1016/j.ijfoodmicro.2014.02.026
Tan M, Jiang X (2005) Norovirus and its histo-blood group antigen
receptors: an answer to a historical puzzle. Trends Microbiol
13(6):285293. doi:10.1016/j.tim.2005.04.004
Thisted Lambertz S, Ballagi-Pordany A, Lindqvist R (1998) A mimic as
internal standard to monitor PCR analysis of food-borne pathogens.
Lett Appl Microbiol 26(1):911
Tian P, Mandrell R (2006) Detection of norovirus capsid proteins
in faecal and food samples by a real time immuno-PCR
method. J Appl Microbiol 100(3):564574. doi:10.1111/j.
1365-2672.2005.02816.x
Tian P, Bates AH, Jensen HM, Mandrell RE (2006) Norovirus binds to
blood group A-like antigens in oyster gastrointestinal cells. Lett Appl
Microbiol 43(6):645651. doi:10.1111/j.1472-765X.2006.02010.x
Tu S, Reed S, Gehring A, He Y (2011) Simultaneous detection of
Escherichia coli O157:H7 and Salmonella Typhimurium: the use
of magnetic beads conjugated with multiple capture antibodies.
Food Anal Methods 4:357364
Urbanucci A, Myrmel M, Berg I, von Bonsdorff CH, Maunula L (2009)
Potential internalisation of caliciviruses in lettuce. Int J Food
Microbiol 135(2):175178. doi:10.1016/j.ijfoodmicro.2009.07.036
Wilde J, Eiden J, Yolken R (1990) Removal of inhibitory substances from
human fecal specimens for detection of group A rotaviruses by
reverse transcriptase and polymerase chain reactions. J Clin
Microbiol 28(6):13001307
Wolffs P, Knutsson R, Sjoback R, Radstrom P (2001) PNA-based light-
up probes for real-time detection of sequence-specific PCR prod-
ucts. Biotechniques 31(4):766, 769771
Wolffs P, Knutsson R, Norling B, Radstrom P (2004) Rapid quantifica-
tion of Yersinia enterocolitica in pork samples by a novel sample
preparation method, flotation, prior to real-time PCR. J Clin
Microbiol 42(3):10421047
Wolffs P, Norling B, Hoorfar J, Griffiths M, Radstrom P (2005)
Quantification of Campylobacter spp. in chicken rinse samples by
using flotation prior to real-time PCR. Appl Environ Microbiol
71(10):57595764. doi:10.1128/AEM.71.10.5759-5764.2005
Woudstra C, Skarin H, Anniballi F, Auricchio B, De Medici D, Bano L,
Drigo I, Hansen T, Lofstrom C, Hamidjaja R, van Rotterdam BJ,
Koene M, Bayon-Auboyer MH, Buffereau JP, Fach P (2013)
Validation of a real-time PCR based method for detection of
Clostridium botulinum types C, D and their mosaic variants C-D
and D-C in a multicenter collaborative trial. Anaerobe 22:3137.
doi:10.1016/j.anaerobe.2013.05.002
Zhang G, Brown EW, Gonzalez-Escalona N (2011) Comparison
of real-time PCR, reverse transcriptase real-time PCR, loop-
mediated isothermal amplification, and the FDA conventional
microbiological method for the detection of Salmonella spp.
in produce. Appl Environ Microbiol 77(18):64956501. doi:
10.1128/AEM.00520-11
Zhao T, Zhao P, Doyle MP (2012) Detection and isolation of Yersinia
pestis without fraction 1 antigen by monoclonal antibody in foods
and water. J Food Prot 75(9):15551561. doi:10.4315/0362-028X.
JFP-11-514
Zuo H, Xie Z, Ding X, Zhang W, Yang J, Fan X, Poms R, Pei X (2011) A
novel magnetic xapture-multiplex PCR assay for the simultaneous
detection of three foodborne pathogens. Qual Assur Saf Crops Foos
3:212220