1846 Junge et al.: Journal of AOAC International Vol. 94, No.

6, 2011
FOOD BIOLOGICAL CONTAMINANTS

BIOTECON Diagnostics foodproof E. coli O157 Detection Kit, 5 Nuclease for E. coli O157 in Combination with foodproof ShortPrep II Kit
Performance Tested MethodSM 100601 Abstract The method describes the detection of Escherichia coli O157 in food. The method is based on real-time PCR using hydrolysis probes (5 Nuclease). This advanced PCR method was designed to reduce the time necessary to achieve results from PCR reactions and enable the user to monitor the amplification of the PCR product simultaneously in real time. After DNA isolation using the BIOTECON foodproof ShortPrep II Kit designed for the rapid preparation of E. coli O157 DNA for direct use in PCR, the real-time detection of E. coli O157 DNA is carried out using the foodproof E. coli O157 Detection Kit. The kit provides primers and hydrolysis probes for sequence-specific detection, convenient premixed reagents, and controls for reliable interpretation of results. For repeatability studies three different foods (egg salad, large bockwurst/frankfurter, and apple juice) were analyzed, chosen from the 15 food groups recommended by the AOAC Research Institute for E. coli O157 detection. From each food, 20 samples were inoculated with a low level (110 CFU/25 g) and 20 samples with a high level (1050 CFU/25 g) of E. coliO157. Additionally, five nonspiked samples were prepared from each food. Depending on the matrix, the food samples were examined with the test kits and compared with the cultural methods according to the U.S. Food and Drug Administration Bacteriological Analytical Manual or the U.S. Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook. Participants Method Authors Benjamin Junge, Cordt Grnewald, and Kornelia Berghof-Jger BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam, Germany
Submitted for publication March 25, 2011. The method was independently tested, evaluated, and certified by the AOAC Research Institute as a Performance Tested MethodSM. See http://www.aoac.org/testkits/steps.html for information on certification. Corresponding authors e-mail: bjunge@bc-diagnostics.com DOI: 10.5740/jaoacint.11-097

Submitting Company BIOTECON Diagnostics GmbH, Hermannswerder Haus17, 14473 Potsdam, Germany Reviewers Thomas Hammack and Yi Chen U.S. Food and Drug Administration, 5100 Paint Branch Pkwy, College Park, MD 20740 Introduction Principle The method describes the detection of E. coli O157 in food. The method is based on real-time PCR. The foodproof ShortPrep II Kit is used for the isolation of E. coli O157 DNA from enriched food samples. The foodproof E. coli O157 Detection Kit detects E. coli O157 specific DNA by means of real-time PCR using 5 Nuclease- (TaqMan )-based instruments. General Information Target organisms.Although most strains of the species Escherichia coli are harmless and live in the intestines of healthy humans and animals, strains of serotype O157 (with a few exceptions) produce a powerful toxin and can cause severe illness. Especially E.coli O157:H7 but also other E. coli O157 serotypes are an emerging cause of foodborne illness. An estimated 73000 cases of infection and 61 deaths occur in the United States each year. Infection often leads to bloody diarrhea, and occasionally to kidney failure. Most illness has been associated with eating undercooked, contaminated ground beef (1). The LightCycler foodproof E. coli O157 Detection Kit detects all E. coli of serotype O157 including the O157:H7 serotype. Matrixes.Three food groups recommended by the AOAC Research Institute for E. coli O157 were tested: egg salad, large bockwurst/frankfurter, and apple juice. Summary of validated performance claims.For the repeatability study three different food samples out of the 15 food groups were tested with the foodproof E.coliO157 Detection Kit, 5 Nuclease in combination with the foodproof ShortPrep II Kit. The food samples were tested in correlation with the cultural methods

Junge et al.: Journal of AOAC International Vol. 94, No. 6, 2011 1847 Table 2. Reagents of the foodproof E. coli O157 detection kit
Component foodproof E. coli O157 Master Mix (vial 1) foodproof E. coli O157 Enzyme Solution (vial 2) foodproof E. coli O157 Internal Control (vial 3) Total volume Volume, mL 18.0 1.0 1.0 20.0

Table 1. Repeatability study: E. coli O157 strains and foods

Food Egg salad Bockwurst Apple juice
a

BCDa Strain No. 14178 14226 14244

Serotype O157:HO157:H7 O157:H7

BCD = BIOTECON Diagnostics GmbH.

according to U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM) and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS).
Summary of Results

(b) foodproof ShortPrep II Kit.Reagent 1. Reaction tubes with 800 L ready-to-use foodproof ShortPrep II Lysis Reagent. Reagent 2.Bottle with 20mL foodproof ShortPrep II Resuspension Reagent. Additional Supplies and Reagents (a) Microbiology laboratory equipment.e.g., Nucleasefree, aerosol-resistant pipet tips; pipets with disposable, positive-displacement tips; sterile reaction (Eppendorf) tubes for preparing master mixes and dilutions; LightCycler capillaries, Roche Diagnostics (Mannheim, Germany) Product No. 1 909 339; LightCycler Color Compensation Kit, Roche Diagnostics Product No. 2 158 850; optional: LightCycler Carousel Centrifuge, Roche Diagnostics Product No. 2 189 682 or 3 030 512; 96-well plates for Mx3005P, e.g., 4titude Cat. No.4ti-0710/B. (b) Media and reagents for E. coli O157.BAM, Chapter 4a (July 2009) were used. (c) Media and reagents for E. coli O157.USDA FSIS, MLG 5.04 and MLG 5A.01 for meat (January 2008) were used. (d) Equipment and materials for E. coli O157 BAM assay.Balance >2 kg with 0.1 g sensitivity; stomacher; incubators, 35 0.5C and 44 1C; Petri dishes 20 150 mm; Pasteur pipets; pH test paper, range 6.08.0; screw cap stomacher strainer; and 400 sterile filter bags or equivalent. (e) Equipment and materials for E. coli O157 EN ISO 16654 assay.Autoclave; incubators; water bath; pH analyzer; measuring cylinder; pipets, 25, 10, and 1 mL; tubes, 16 125 mm or other appropriate sizes; screw cap stomacher strainer; 400 sterile filter bags or
Table 3. Interpretation of results using two different fluorescence channels
E. coli O157 channel FAM Positive Negative Positive Negative Internal control channel VIC/HEX Positive Positive Negative Negative Result interpretation Positive Negative Positive Invalid

The repeatability study/method comparison study was accomplished with three different food matrixes. The results of the three low and high inoculated foods showed a very high correlation with the PCR method and the cultural FDA-BAM or USDA/FSIS methods. All uninoculated foods were negative. The inclusivity and exclusivity studies gave the expected results on both real-time PCR instruments (LC 480 and Mx3005P). No false positives or false negatives occurred. The specificity was confirmed at 100%. Materials and Method Test Kit Information (a) Kit names.foodproof E. coli O157 Detection Kit, 5 Nuclease foodproof ShortPrep II Kit. (b) Cat. Nos.Cat. No. R 302 10; foodproof E.coli O157 Detection Kit, 5 Nuclease. Cat. No. S 400 02; foodproof ShortPrep II Kit. Test Kit Reagents (a) foodproof E. coli O157 Detection Kit, 5 Nuclease. Reagent 1.foodproof E. coli O157 Master Mix, ready-to-use primer, and hydroysis probes for E. coli O157 DNA and the E. coli O157-specific internal control (yellow cap). Reagent 2.foodproof E. coli O157 Enzyme Solution, contains Taq DNA polymerase and uracil-DNA glycosylase (heat-labile) for prevention of carryover contamination (red cap). Reagent 3.foodproof E. coli O157 internal control, contains a stabilized solution of plasmid DNA and a yellow dye for better visualization (white cap). Reagent 4.foodproof E. coli O157 control template, contains a stabilized solution of plasmid DNA for use as a PCR run positive control (purple cap). Reagent 5.H2O, PCR-grade, contains nuclease-free, PCR-grade H2O (colorless cap).

1848 Junge et al.: Journal of AOAC International Vol. 94, No. 6, 2011 Table 4. Results of the internal comparison study with three food matrixes tested by PCR and microbiologically according to the FDA-BAM or USDA/FSIS methods
Food Egg salada No. of samples 20 20 5 Bockwurst
b

Inoculation level (determination via MPN), cells/25 g 1.1 11.5 0.2 5.8 0.4 5.8

Inoculation level, cells/1 g 0.04 0.46 0.007 0.23 0.015 0.23

PCR 5 16 0 6 10 0 7 14 0

Cultural confirmation 5 16 0 6 10 0 7 14 0

FDA-BAM or USDA/FSIS 6 15 0 6 10 0 7 14 0

20 20 5

Apple juicea

20 20 5

a b

Food matrixes tested according to the FDA-BAM method. Food matrix tested according to the USDA/FSIS method.

equivalent; apparatus for immunomagnetic separation, inoculating loops; inoculating needle; microscope slides; needle, scalpels, chisels, knives, scissors, spatulas, forceps, disposable or reusable dishes, pans or trays; Petri dishes, 90 to 140 mm; and vortex mixer.
Apparatus

(a) LightCycler 480 real-time PCR system.Roche Diagnostics. The LightCycler 480 real-time PCR system is a high throughput gene quantification or genotyping real-time PCR platform with exchangeable blocks for 96 and 384 samples in multiwell plates. It offers enhanced throughput, compatibility with automation, and maximum flexibility regarding hardware and software. The LightCycler 480 real-time PCR system can complete a 96 well PCR run in 40 min. The LightCycler 480 real-time PCR system is a multiwell-plate based real-time PCR platform that is used for highly accurate qualitative and quantitative detection of nucleic acids and genotyping. Building on the benefits of Roches capillary-based LightCycler 1.5 and 2.0 systems, it goes one step further in offering enhanced throughput, compatibility with automation
Table 5. 2 values of the high inoculated food samples (1050 CFU/25 g)
No. of PCR positive/reference method negative (a) 1 0 0 No. of PCR negative/reference method positive (b) 0 0 0

equipment, and maximum flexibility regarding hardware and software. (b) Mx3005P QPCR system.Agilent Technologies (Santa Clara, CA). The Mx3005P system is among the most reliable and trusted QPCR instruments available, with a long record of citation in peer-reviewed journals. Offering unmatched flexibility and reliability, the system is ideal for a wide variety of applications and chemistries, including but not limited to gene expression analysis, microarray data validation, single nucleotide polymorphism genotyping, pathogen detection, DNA methylation analysis, and chromatin immuneprecipitation studies. Highly reproducible results are the product of the Mx3005Ps single light source, single detector precision optic scanning design, providing uniform excitation and detection, coupled with the trusted Peltier-based thermal system, which ensures uniform ramping and thermal accuracy. (c) Pentium computer with LightCycler and MxPro software.For analysis and storage of results. (d) Bench-top microcentrifuge.Rotor size to fit 2.0 mL reaction tubes (e.g., Eppendorf Centrifuge 5415C). (e) Bench-top vortexing unit.To mix/vortex samples during foodproof ShortPrep procedure (e.g., Heidolph, REAX 2000, Germany). (f) Heating unit.e.g., Eppendorf ThermoStat plus 5352 000.0. Standard Reference Materials For each food matrix one different E. coli O157 strain was used. Table 1 shows the foods and the applied E. coli O157 strains with their origin and properties.

Food Egg salad Bockwurst Apple juice

2 0 0 0

Junge et al.: Journal of AOAC International Vol. 94, No. 6, 2011 1849

Table 6. 2 values of the low inoculated food samples (110 CFU/25 g)

No. of PCR positive/reference method negative (a) 0 0 0 No. of PCR negative/reference method positive (b) 1 0 0

Food Egg salad Bockwurst Apple juice

2 0 0 0

Standard Solutions (a) Pre-enrichment media according to FDA-BAM (Feng, P., September 2002, BAM Online, Chapter 4a: Diarrheagenic E. coli O157). (b) Pre-enrichment media according to USDA/FSIS (Microbiology Laboratory Guidebook 5.04 for meat). General Preparation The heating unit was turned on and temperature was set to 95100C. The container was filled with ice for storage of foodproof ShortPrep II kit samples. All sample tubes were labeled for identification of samples. The LightCycler 480 and Mx3005P instruments and attached PCs were turned on. Sample Preparation Pre-enrichment.To 25 g of the food sample 225mL pre-enrichment media was added according to the FDABAM and USDA/FSIS instructions. Analysis (a) foodproof ShortPrep II kit (Cat. No. S 400 02), preparation/extraction of DNA from food enrichment. In order to collect the lysis reagent at the bottom of the tube, the reaction tube with the ready-to-use reagent was centrifuged at 500 g for 3060 s. The enriched culture was shaken gently and allowed to settle for 510 min; 200L of the sample (supernatant) was then transferred to the reaction tube containing the ready-to-use lysis reagent. The reaction tube had to be firmly closed. The lysis reagent and sample were mixed by vortexing. The reaction tube was centrifuged in a bench-top microcentrifuge for 5 min at 8000 g. The supernatant was discarded by pipetting and inactivated appropriately. A 200L volume of resuspension reagent was added. The tube was placed in the cell disruption unit and turned on for 8 min for mechanical disruption. The reaction tube was incubated in the heating unit for 10 min at 95100C. The reaction tube was carefully removed from the heating unit, using forceps, as the tube was hot. The tube was allowed to sit for 1 min, then the sample was mixed for

2 s by vortexing. After centrifugation at 13000 g for 1min at room temperature, the supernatant contained the extracted DNA. The prepared sample was stored at 4C if the PCR followed immediately; otherwise it was stored at 20C. (b) foodproof E. coli O157 Detection Kit, 5 Nuclease (Cat. No. R 302 10), preparation of the PCR mix.A 25L standard reaction was prepared as described below. Note: Gloves must be used when handling the PCR vessels. The solutions were thawed and, for maximal recovery of contents, vials were briefly centrifuged in a microcentrifuge before opening. The solutions were mixed carefully but thoroughly by pipetting up and down. In a reaction tube (0.52.0 mL, depending on the number of reactions), the PCR mix was prepared by adding the following components in the order mentioned below. The volumes indicated are based on a single 25 L standard reaction. The PCR mix was prepared by multiplying the amount in the Volume column by the number of reactions to be cycled plus one or two additional reactions to cover pipetting losses (Table 2). Instructions (1) Mix carefully but thoroughly by pipetting up and down. Do not vortex. Pipet 20 L PCR mix into each PCR vessel. For the samples of interest, add 5 L sample DNA. For the negative control, add 5 L H2O, PCRgrade (vial 5, colorless cap). For the positive control, add 5L foodproof E. coli O157 control template. (2) Seal the PCR vessels accurately with optical caps or foil. Briefly centrifuge the PCR vessels in a suitable centrifuge. Cycle the samples as described below: Preincubation.1 cycle. Step 1.37C for 4 min. Step 2.95C for 5 min. Amplification.50 cycles. Step 1.95C for 5 s. Step2 (fluorescence detection in Step 2).60C for 60s. Interpretation and Test Result Report Description of interpretation procedure and how results are reported.The amplification of DNA of E. coli O157 was analyzed in the fluorescence channel suitable for FAM-labeled probes detection. The specific amplification of the internal control was analyzed in the fluorescence channel suitable for VIC/HEX. The results
Table 7. Distribution of inclusivity strains
Serovar E. coli O157:H7 E. coli O157:HE. coli O157:H16 E. coli O157, H unknown No. strains 38 19 2 1

1850 Junge et al.: Journal of AOAC International Vol. 94, No. 6, 2011 Table 8. Inclusivity strain number and origin
Strain No. (internal) 4735 4738 4946 4948 5579 5580 5854 5855 7840 7842 7844 7848 7851 7852 7854 7855 7875 8275 8325 12503 12507 12518 12538 14173 14174 (ATCC 43895) 14175 14176 14177 14178 14190 14200 14211 14226 14227 14240 14241 14242 14243 14244 14245 14246 14247 Serotype O157:HO157:H7 O157:H7 O157 O157:H7 O157:H7 O157:H7 O157:HO157:HO157:HO157:HO157:HO157:HO157:HO157:H7 O157:H7 O157:HO157:H7 O157:H7 O157:HO157:HO157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:HO157:H16 O157:HO157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 Origin/source Unknown Human feces Human Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Milk Sausages Minced meat (cattle) Bovine feces Bovine feces Hamburger Raw milk Human feces Ground beef Milk Human feces Minced meat (cattle) Minced meat (cattle) Bovine feces Bovine feces Bovine feces Bovine feces Human feces Human feces Human feces Human feces Human feces Human feces

Table 8. (continued)
Strain No. (internal) 14248 14249 14250 14251 14252 14253 14254 14255 14256 14257 14258 (NCTC 12079) 14259 14260 14261 14262 14263 14264 14265 (NCTC 12080) Serotype O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:HO157:HO157:HO157:H16 O157:HO157:HO157:HOrigin/source Human feces Human feces Human feces Human feces Human feces Human feces Human feces Salami Ground beef Ground beef Human feces Human feces Sausages Ground beef Ground beef Raw milk Intestine, sheep Human feces

were compared from channel FAM (E. coli O157) and channel VIC/HEX (internal control) for each sample, and the results were interpreted as described in Table 3. Summary of Results Internal Comparison Study The alternative PCR method was performed on two different real-time PCR instrumentsLightCycler 480 and Mx3005Pwith the same results (Table 4). The foodproof E. coli O157 detection kit in combination with the foodproof ShortPrep II kit successfully detected low and high numbers of E. coli O157 in food samples. All uninoculated food samples were negative; no false-positive results occurred. The results of the three low and high inoculated food samples showed a very high correlation with the PCR method and the cultural FDA-BAM or USDA/FSIS methods. Calculation of performance indicators.The four performance indicators for qualitative methods are sensitivity, specificity, false-positive rate, and falsenegative rate. The definitions of performance indicators are as follows: Sensitivity.Sensitivity or inclusivity is the ability of the method to detect E. coli O157 from a wide range of

 Table 9. Exclusivity strains

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Organism Citrobacter koseri Cronobacter sakazakii

Junge et al.: Journal of AOAC International Vol. 94, No. 6, 2011 1851

Strain No. (internal) Strain No. (external) 4958 4955 15136 12509 12512 12504 12505 12506 14206 12511 5856 5853 5858 12514 5852 5851 12508 5647 5648 5857 12515 5849 12502 12510 12513 5611 8930 4957 14151 2144 DSM 4595 DSM 4485 DSM 30054 LM 1126 LM 1364 LM 841 LM 872 LM 1046 1608 LM 1328 H 2459/96/1 H 73/96/1 H 2955/96/1 LM 1394 H 509/95 H 774/89 LM 1119 EH VUB 60 7828/95 H 2947/96/1 LM 1398 H 946/87/1 LM 680 LM 1247 LM 1389 DSM 4564 DSM 30163 DSM 30102 2627/00 DSM 4782

Strain origin/source Throat Childs throat Spinal fluid Meat (lamb) Minced meat (cattle) Beef Raw milk Minced meat (cattle) Minced meat (cattle) Raw milk cheese Unknown Unknown Unknown Minced meat (cattle) Unknown Unknown Meat (lamb) Unknown Unknown Unknown Minced meat (cattle) Unknown Milk Meat (lamb) Minced meat (cattle) Human wound Human clinical isolate Water Human stool Unknown

Enterobacter cloacae subsp. cloacae E. coli O7:H- (STEC) E. coli O8:H- (STEC) E. coli O8:H27 (STEC) E. coli O17:H- (STEC) E. coli O22:H- (STEC) E. coli O22:H8 (STEC) E. coli O23:H15 (STEC) E. coli O26:H- (STEC) E. coli O26:H11 (STEC) E. coli O26:H11 (STEC) E. coli O46:H- (STEC) E. coli O48:H21 (STEC) E. coli O55:H- (STEC) E. coli O84:H21 (STEC) E. coli O101:H9 (STEC) E. coli O103:H2 (STEC) E. coli O103:H2 (STEC) E. coli O104:H12 (STEC) E. coli O111:H2 (STEC) E. coli O138:H8 (STEC) E. coli Ont:H- (STEC) E. coli Orauh:H23 (STEC) Escherichia vulneris Hafnia alvei Klebsiella pneumoniae subsp. pneumoniae Salmonella enterica subsp. enterica (Enteritidis) Shigella flexneri

strains in an as-small-as-possible amount of CFU/25 g food sample. Sensitivity rate (p+) for a food type and inoculation level is defined as the probability that a method, alternative or reference, will classify a test sample as positive, given that a test sample is a known positive. A known positive refers to the confirmation of inoculated analyte. Sensitivity rate is defined as the total number of confirmed positive test portions by the method divided by total number of confirmed positive test portions by both the alternative and reference methods. Specificity.Specificity or exclusivity is the lack of interference in the alternative method from a relevant range of nontarget strains, which are potentially cross-reactive. Specificity rate (p+) for a food type and inoculation level is defined as the probability that a method, alternative or reference, will classify a test sample as negative, given

that a test sample is a known negative. A known negative refers to a confirmed negative test portion. Specificity rate is defined as the total number of confirmed negative test portions by the method divided by total number of confirmed negative test portions by both the alternative and reference methods. Repeatability.The repeatability is within-laboratory precision, designated sr, or the closeness of agreement between successive and independent results obtained by the same method on identical test material, under the same conditions (e.g., apparatus, operator, laboratory, and incubation time). Stability.Stability is defined as consistent detection results of Salmonella without influence of different production lots or shelf life. In the following the combined results of the whole

1852 Junge et al.: Journal of AOAC International Vol. 94, No. 6, 2011

repeatability study (in-house and independent validation studies) for these four performance indicators are provided: Low inoculation level (110 CFU/25 g).Sensitivity rate (p+) = 18/19 = 0.95; specificity rate (p) = 42/41 = 1.02; false-positive rate (pf+) = 0/41 = 0; false-negative rate (pf) = 1/20 = 0.05. High inoculation level (1050 CFU/25 g). Sensitivity rate (p+) = 40/39 = 1.03; specificity rate (p) = 20/21 = 0.95; false-positive rate (pf+) = 1/22 = 0.045; false-negative rate (pf) = 0/39 = 0. Test of significance difference (2).The alternative method must be statistically equivalent to the reference method for each food matrix and each inoculation level. Equivalence is measured using the Chi-square (2) methodology. For qualitative methods the McNemar test is conducted. A Chi-square value <3.84 indicates that the hypothesis that the test method and reference method are equivalent could not be rejected at the 5% level of confidence. This criterion must be satisfied for each level of each food type. Chi-square, as defined by McNemar, is:

real-time PCR instruments, the LightCycler 480 and the Stratagene Mx3005P, that organisms other than E. coli O157 are not detected with the foodproof E. coli O157 Detection Kit, 5 Nuclease. Thirty strains of nontarget organisms were tested, including E. coli of other serotypes as well as strains of the same microbiological environment. The suitability of the extracts for PCR are shown with a consensus-PCR system (Table 9). The foodproof E. coli O157 Detection Kit was specific for E. coli of serotype O157 on both real-time PCR instruments. No false-positive results occurred. Conclusions For this method extension a repeatability study/method comparison with three different food matrixes was accomplished. Moreover the inclusivity and exclusivity of the real-time PCR system were examined with a wide spectrum of different isolates. Therefore the BIOTECON Diagnostics foodproof E. coli O157 Detection Kit in combination with the foodproof ShortPrep II kit was tested on two different real-time PCR instruments, the LightCycler 480 System from Roche Diagnostics and the Mx3005P from Agilent/Stratagene. The repeatability study and the inclusivity and exclusivity studies gave the expected results. No deviations occurred, and all results were within the expected range. References
(1) Centers for Disease Control and Prevention, http://www. cdc.gov/ncidod/dbmd/diseaseinfo/escherichiacoli_g.htm (2) U.S. Food and Drug Administration (2011) Bad Bug BookEscherichia coli O157, http://vm.cfsan.fda. gov/~mow/chap15.html (3) Karmali, M.A. (1989) Clin. Microbiol. Rev. 2, 1538. doi:10.1006/fmic.1997.0134 (4) Scheu, P.M., Berghof, K., & Stahl, U. (1998) Food Microbiol. 15, 1331 (5) Grant, M.A. (2004) Appl. Environ. Microbiol. 70, 12261230. doi:10.1128/AEM.70.2.1226-1230.2004 (6) U.S. Food and Drug Administration (2011) Bacteriological Analytical Manual, 8th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, pp 4.014.29, http://www.cfsan.fda.gov/~ebam/bam-4a.html (7) Richter, H., Klie, H., Timm, M., Gallien, P., Perlberg, K.W., Teufel, P., Protz, D., & Steinrck, H. (1998) Infektionsepidemiol. Forsch. II/98, 36 (8) EN ISO 16654:2001, Horizontal Method for the Detection of E. coli O157, Beuth Publishing, Berlin, Germany, pp 114

where a = test samples positive by the alternative method and negative by reference method, b = test samples negative by the alternative method and positive by reference method. Tables 5 and 6 show the Chi-square results for each food type, each table for one inoculation level. The criterion of a Chi-square value <3.84 was achieved for each level of each food type. Specificity Inclusivity.The objective was to verify on both real-time PCR instruments, the LightCycler 480 and the Stratagene Mx3005P, that different strains of E. coli of serotype O157 are detected with the foodproof E. coli O157 Detection Kit, 5 Nuclease. Sixty strains with known serotype O157 were tested with the foodproof E. coli O157 Detection Kit. The tested strains belonged to the serovars listed in Tables 7 and 8. The foodproof E. coli O157 Detection Kit detected various isolates of E. coli of serotype O157 with both real-time PCR instruments. No false-negative results occurred. Exclusivity.The objective was to verify on both

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