PCR: design delivers success

Release Date: 
Wednesday, April 4, 2018 - 14:45

Polymerase Chain Reaction (PCR) has gained wide acceptance as a pathogen detection tool in the food industry, and the popularity of the method continues to grow. Two choices in PCR technology are available for the industry to choose from end-point and real-time PCR (RT-PCR).

In end-point PCR, primers or short oligonucleotides are designed to flank the target sequence which is amplified during the PCR reaction. The amount of PCR product (amplicon) produced during the PCR is detected using a non-specific double strand DNA (dsDNA) binding dye.

Traditionally, the amplicon was visualised using agarose gel electrophoresis. But fluorescent dyes such as SYBR Green are now used to measure the accumulated PCR product at the end of PCR without the need to run a gel and thus keeping the system closed. Typically, this is achieved by performing a heating step during which the double stranded (ds)DNA come apart releasing the fluorescent dye and consequently resulting in a decrease in fluorescent signal. The amount and temperature at which the fluorescent signal changes correspond to the strength and type of PCR product generated during the PCR event.

In comparison to end-point PCR, real-time PCR (RT-PCR) detects the increasing amount of amplicon in real-time after each PCR cycle by measuring the fluorescence signal. The changes in fluorescence intensity with each PCR cycle are monitored in an amplification plot, from which the cycle threshold (Ct) is calculated.

The Ct value, also referred to as the crossing point (Cp), is inversely correlated to the amount of DNA present in the sample, and is used to measure the amount of amplicon produced in real-time. This measurement can be used for absolute quantification through a standard curve or relative quantification by comparison with known amount of a reference target.

The fluorescent signal in RT-PCR is generated by a fluorescently labelled, target-specific DNA probe which is included in addition to the PCR primers. A quencher present in the probe suppresses the fluorescence in the absence of the target sequence, but is released when the target is present. RT-PCR assays provide an option of multiplexing to include many targets in a single assay. Probes for different targets are designed with reporter dyes that emit fluorescence at different emission wavelengths. Together with a unique set of primers, amplicons generated for each target emit a unique fluorescent signal.

The advantages of RT-PCR are added specificity due to the probe and the ability to multiplex. However, these advantages should be weighed against the complexity of probe design and the cost of the probe which is expensive due to the fluorescent reporter dye and quencher. On the other hand, endpoint PCR affords a cheaper PCR option and simpler design with more reliance on the primers for specificity.

Regardless of the PCR technology selected, the key to the success of a PCR assay in food testing lies in its design and its validation. For an endpoint assay, the primers should be carefully designed to be selective for the target and to meet inclusivity/ exclusivity criteria. For a RT-PCR assay, in addition to the primers, the design of the probe is critical to the selectivity of the assay. For both assays, initial primers/probe design is done by using bioinformatics, followed by wet laboratory testing with bacterial strains to confirm specificity and selectivity.

Furthermore, to achieve robustness, it is imperative to optimise amounts of other critical PCR components including DNA polymerase, nucleotides and salts. The addition of an internal positive control as an indicator of assay performance provides increased reliability and confidence in results. Finally, validating the performance of the PCR chemistry on a wide range of appropriate matrices is essential to ensure the success of the assay and monitoring food safety!

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