Polymerase Chain Reaction (PCR) has gained widespread acceptance as a tool for detecting pathogens in the food industry, and the method continues to grow in popularity. Two options within PCR are available to the industry: endpointPCR andPCR. Read on to learn about the differences and how they work. This is the second part of our article series on PCR.
End-pointPCR
InPCR , primers or short oligonucleotidesPCR to flank the target sequence that is amplified during PCR. The amount of PCR(amplicon) produced during PCR using a non-specific double-stranded DNA (dsDNA) binding dye. Traditionally, the amplicon was visualized using agarose gel electrophoresis. However, fluorescent dyes such as SYBR Green are now used to measure the accumulated PCR at the end of PCR having to run a gel, thereby keeping the system closed. This is typically achieved by performing a melting step during which the double-stranded (ds)DNA unravels and releases the fluorescent dye, consequently resulting in a decrease in the fluorescent signal. The magnitude and temperature at which the fluorescent signal changes correspond to the strength and type of PCR generated during PCR.
Real-timePCR
Compared toPCR ,PCR PCR PCR increasing amount of amplicon in real time after each PCR by measuring the fluorescence signal. Changes in fluorescence intensity with each PCR are monitored in an amplification curve, from which the cycle threshold (Ct) is calculated. The Ct value, also known 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 measure can be used for absolute quantification via a
standard curve or relative quantification by comparison with a known amount of a reference target.
The fluorescent signal inPCR by a fluorescently labeled,
target-specific DNA probe included in addition to PCR. A quencher present in the probe suppresses fluorescence in the absence of the target sequence but is released when the target is present.PCR allow for multiplexing to include multiple 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, the amplicons generated for each target emit a unique fluorescent signal. The advantages ofPCR enhanced 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,PCR offersPCR more affordable PCR and a simpler design that relies more on the primers for specificity.
Summary
Regardless of the PCR chosen, the key to the success of a PCR in food testing lies in its design and validation. For an endpoint assay, the primers must be carefully designed to be selective for the target and to meet inclusion/exclusion criteria. For aPCR, in addition to the primers, the design of the probe is critical to the assay’s selectivity. For both assays, initial primer/probe design is performed using bioinformatics, followed by wet lab testing with bacterial strains to confirm specificity and selectivity. To achieve robustness, it is also absolutely essential to optimize the amounts of other critical PCR, 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 the results. Finally, it is important to validate the performance of PCR across a broad spectrum of suitable matrices to ensure the success of the assay and monitor food safety!