PCR: What, why and how?

Release Date: 
Sunday, February 4, 2018 - 14:15

Advancements in rapid molecular methods have revolutionised pathogen detection in food safety. While culture methods still serve as the ‘gold standard’ in the industry, newer technologies promise to deliver equal detection sensitivity in a significantly shorter time.

This series of articles will discuss one of the most popular rapid methods measuring nucleic acids.

Nucleic acids, including DNA and RNA are biomolecules that are an integral part of all living cells. This ubiquitous nature makes them excellent indicators for the presence of life forms, including pathogenic microorganisms.

The Polymerase Chain Reaction (PCR) is an amplification technique that generates thousands of copies of a targeted DNA region in a few hours. This makes PCR a powerful tool for detection of specific bacteria such as pathogens.

The basic components of PCR include primers, the DNA polymerase enzyme, nucleotides, specific buffering ions and a target DNA template. Primers are short fragments of DNA that are complementary to specific segments of the target DNA.

Primers are designed to target unique regions of the DNA, lending high specificity to PCR. The reaction begins with the ‘denaturation’ step at high temperatures, usually 95°C, when the double stranded DNA template is separated into two individual strands.

This is followed by a drop in the temperature, the cooling allows for the ‘annealing’ or binding of the primers to its complementary region on the DNA. The enzyme DNA polymerase then performs the ‘extension’ step by attaching near the end of primer and begins adding nucleotides resulting in the PCR product called as ‘amplicon’.

Several copies of amplicon are created as the three steps repeat over a set number of cycles. The amplicons produced during PCR can be detected by stains such as ethidium bromide or fluorescent dyes such as SYBR Green. The detection can occur at the end of the reaction or during the reaction using a specialised PCR technique called Real-time PCR.

PCR based pathogen detection assays provide various advantages over the culture confirmation methods.

• Rapid detection:

Results are obtained in 1-2 hours compared to 3-5 days by cultural methods.

• High sensitivity:

PCR can detect a single copy of DNA.

• High specificity and selectivity:

PCR assays can be designed to be highly specific and selective for species and strains.

• Quantitation:

Allows enumeration of bacteria in the sample.

Unlike cultural methods, PCR does not depend on the physiological state of the cell. The method performs with equal efficiency even in cases where

culturing cells may be challenging. Conversely the test cannot differentiate living from dead cells without some form of sample preparation.

Some samples may contain materials that interfere with the assay and results, and solutions to these challenges will be addressed in following articles.

PCR detection systems have been used for industrial food applications for >20 years and has seen advances in instrumentation and software for data analysis, reagents and workflow leading to improvements in robustness, ease of use and time to result. These attributes have undeniably made PCR a front runner in rapid pathogen detection methods.

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contactus@hygiena.com

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