PCR-based foodborne pathogen detection methods & applications in the food industry

FS 362

Review
What is polymerase chain reaction (PCR) and what is the purpose of this reaction? What role does temperature play in PCR? What role do primers play in PCR? Characteristics of well-designed primers. 5 ways to optimize a PCR reaction.

Today
PCR platforms Advantages/Disadvantages of PCR detection systems Characteristics of high quality assays Good Laboratory Practices

The Central Dogma

DNA
Molecular methods
DNA replication Transcription

mRNA
Translation

Classical methods

Protein/Enzymes
Post-translation

Toxins & other metabolites

DNA-based detection by PCR

DNA

Detects chromosomal or extra-chromosomal DNA (i.e. plasmids) Advantages:

Disadvantages:

DNA-based detection by PCR

DNA
DNA replication Transcription

mRNA

Examples used in industry

hly assay for Listeria monocytogenes
Listeria spp. v. L. monocytogenes

invA assay for Salmonella

Salmonella v. non-Salmonella

Multiplex for STEC

Detection of multiple genes required to identify strain likely to cause severe disease

Multiplex PCR
Used to target multiple genes in a single test
Advantages More information from a single test Use less reagents/consumables Save on time Disadvantages Requires time for optimization

Single PCR reaction that contains multiple, unique primer sets

Each primer set must amplify fragments of varying sizes specific to different DNA sequences Annealing temperatures for each primer set must be similar in order to work correctly within a single test

Multiplex & STEC

stx1 = 534 bp stx2 = 384 bp eaeA = 255 bp EHEC hlyA = 180 bp

stx2 > stx1 eaeA required hlyA required

RNA-based detection by PCR

Reverse-transcriptase PCR
DNA
Reverse transcriptase

mRNA

cDNA

Protein/Enzymes Toxins and other metabolites

RNA-based detection by PCR

DNA
Reverse transcriptase

Advantages:

mRNA

cDNA

Disadvantages:

Applied Biosystems Salmonella & L. monocytogenes Rapid Detection System

Step 2

Step 3 RT-PCR

Step 4: Analysis

Real-time PCR Works just as conventional PCR does except:

Involves fluorescent dye(s) Data collection throughout the PCR process

Allows detection of target in real time during DNA amplification

PCR Chemistries Used to Detect Foodborne Pathogens

SYBR Green Hydrolysis probe-based (e.g., TaqMan) Molecular Beacon Scorpion

SYBR Green
Binds to double-stranded DNA Light is emitted upon excitation Advantages
Binds to minor groove of dsDNA Prefers G and C base pairs

Optimal excitation/emission wavelengths of 497/520 nm Inexpensive, easy to use Minimal effort to design Works well with a single defined target Binds to any double-stranded DNA Primer dimers Non-specific products Dissociation curve or melt curve must follow the PCR protocol

Disadvantages

Melt curve analysis

EXAMPLE: BAX system for detection of L. monocytogenes or Salmonella

Idaho Technoloogy Inc.

Probe-based PCR: Principle of FRET

Fluorescence Resonance Energy Transfer Quencher dye and reporter dye The excited reporter dye transfers energy to a quencher dye rather than fluorescing Quencher dye must be in close proximity to a reporter dye in order to have a quenching effect
Reporter/donor excitation

Reporter

Quencher

Close proximity

Separation

Hydrolysis probe-based PCR

Also referred to as TaqMan assay Takes advantage of 53 exonuclease activity of Taq

Labeled probe hybridizes to DNA target sequence

Intact probe, the reporter does not fluoresce Taq cleaves the fluorogenic tag from the probe

RT-PCR

Multiplex Real-Time PCR

Cy5 FAM

Texas Red

TET

Probe-based real-time PCR

Advantages
Increased specificity Able to detect multiple targets Allows for quantification Faster than conventional PCR

Disadvantages
Expensive to synthesize

PCR Controls
PCR Controls
Negative Use water as the template Positive DNA that will give the expected result

Internal or amplification control

Monitor for PCR inhibition

Reverse-transcriptase PCR
RT-negative control Sample with no reverse transcriptase enzyme Indicator for level of contaminating genomic DNA

How to evaluate PCR detection methods (i.e., LIFE SKILLS)

What is the target gene?
How/why was it chosen What data are available to support the choice
Virulence data? Sequence data?

What is the target molecule?

DNA? mRNA? rRNA? How is expression of mRNA assured?

What is the assay and assay format?

PCR, other amplification method, detection without amplification (e.g., ACCUPROBETM)

How to evaluate molecular detection methods II

How was the assay validated
Pure cultures, spiked foods, or real samples
What isolates or samples were used What was used as gold standard

- What are the most likely reasons for false positives and false negatives - How does the assay perform in the presence of competitive microflora - What controls are included in the assay
- Internal positive control?

- What does a false negative most likely mean (e.g., does it suggest an avirulent variant)

Good Laboratory Practices for PCR

Separate pre-PCR and post-PCR activities
Perform in physically separate areas Minimize potential contamination with amplified product PCR can be set up in sterile hood to minimize contamination issues. Use new pipette tips when transferring samples Prevent aerosolization when ejecting the tip Change gloves frequently

Use aerosol resistant pipette tips Use powder-free gloves

GLPs for PCR

Spin tubes (e.g., sample tubes) briefly to bring contents away from lid
Avoid touching rim/inside lids of tube to ensure no cross-contamination occurs.

Work slowly do not rush Adequately chill cooling blocks before use. Minimize freeze/thaw cycles for frozen reagents.