Lecture 35 PCR Variations & GLPs

8/13/2019 Lecture 35 PCR Variations & GLPs

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PCR-based foodborne pathogen

detection methods & applications in

the food industry

FS 362


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Review

What is polymerase chain reaction (PCR) andwhat 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.


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DNA-based detection by PCR

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

Advantages:

Disadvantages:

DNA


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DNA-based detection by PCR

DNA

mRNA

DNA replication

Transcription


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Examples used in industry

hlyassay 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


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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


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RNA-based detection by PCR

Reverse-transcriptasePCR

DNA

mRNA

Protein/Enzymes

Toxins and other metabolites

Reversetranscriptase

cDNA


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RNA-based detection by PCR

Advantages:

Disadvantages:

DNA

mRNA

Reversetranscriptase

cDNA


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Applied Biosystems Salmonella &

L. monocytogenes Rapid Detection System

S 2


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Step 2


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Step 3 RT-PCR


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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


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PCR Chemistries Used to Detect

Foodborne Pathogens

SYBR Green

Hydrolysis probe-based (e.g., TaqMan)

Molecular Beacon Scorpion


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Idaho Technoloogy Inc.

Melt curve analysis

EXAMPLE:

BAX system for detection of

L. monocytogenes or

Salmonella


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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 reporterdye in order to have a quenching effect

Close proximity Separation

Reporter/donor

excitation

QuencherReporter


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Hydrolysis probe-based PCR

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

Labeled probe hybridizes toDNA target sequence

Intact probe, the reporterdoes not fluoresce

Taq cleaves the fluorogenictag from the probe


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RT-PCR


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Multiplex Real-Time PCR

TET

FAM

Cy5Texas Red


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Probe-based real-time PCR

Advantages

Increased specificity

Able to detect multiple targets Allows for quantification

Faster than conventional PCR

Disadvantages Expensive to synthesize


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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 reversetranscriptase enzyme

Indicator for level of contaminating genomic DNA


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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, detectionwithout amplification (e.g., ACCUPROBETM)


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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 positivesand false negatives- What does a false negative most likely mean (e.g., does it

suggest an avirulent variant)

- How does the assay perform in the presence ofcompetitive microflora

- What controls are included in the assay- Internal positive control?


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Good Laboratory Practices for PCR

Separate pre-PCR and post-PCR activities Perform in physically separate areas

Minimize potential contamination with amplifiedproduct

PCR can be set up in sterile hood to minimizecontamination issues.

Use aerosol resistant pipette tips Use new pipette tips when transferring samples

Prevent aerosolization when ejecting the tip

Use powder-free gloves Change gloves frequently


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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.

Lecture 35 PCR Variations & GLPs

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