A short commentary on Molecular diagnosis of COVID-19 using RT-PCR

In today's time, the coronavirus pandemic has caused havoc in the world. This is evidenced in the upsurge of research that is being conducted by both public and private sector laboratories. However, there is a lot of curiosity from the public and individuals are asking the question as to how COVID-19 is being investigated in the laboratories? Keeping this in mind, we have tried to provide information in this article about the detection of COVID-19, thus we hope this information will be helpful to understand the detection of the COVID-19 disease using polymerase chain reaction (PCR). Therefore, we will first know what are PCR and the variations thereof between PCR, RT-PCR, qPCR, and RTqPCR and how they work. We will further explain how coronavirus is detected through RT-PCR.

- Rahul K. Chandel*, Manish Kumar, Jagdish C. Kandpal, Balasubramani G L, Monika Mittal, Narendra Singh, Sunil K. Tripathi, Vijay Kumar, Rishab Kapoor, Gurjot Kaur, Neelam, Abhishek Negi, Mohd. Faisal, Alpana Razdan, Gauri Agarwal and Rajat Arora

PCR is a laboratory (in-vitro) technique that is employed Pfor amplifying a targeted portion of DNA. This technique is a cell-free method utilized to synthesize several identical copies of a gene or DNA of interest. Thus, it is a very essential tool in a molecular biology laboratory synonymous to a photocopier as a basic requirement in an office. First, we will outline the requirements for a standard PCR reaction – the following will be needed: DNA polymerase enzyme, magnesium chloride (MgCl2), nucleotides, primers, nuclease free water, target DNA template to be amplified and a PCR machine. The PCR reaction mechanism is simple and starts with an initial Denaturation where the double-stranded DNA is heated to separate the strands (1st step) this allows for the primers to attach to the now single stranded DNA (2nd step). After the primers align to the DNA strand, the DNA polymerase enzyme extends the primers (3rd step) which results into two identical copies of the original DNA template. These three steps are repeated over a series of temperatures and times a single complete process of the three combined is known as an amplification cycle. It is important to optimize every step of the cycle to the DNA template and the set of primers being used. The cycles can be repeated as many times as needed, however, on average a standard PCR is run for around 30 to 40 times after which the amplified product is then analyzed. The polymerase chain reaction is used to amplify a targeted DNA of interest for downstream experimental purposes; and it has also found immense use in detection of pathogenic DNA as well as other applications in genetic testing. PCR is a very sensitive technique as tiny volumes of a single reaction are required, as such it is recommended that a master mix is prepared if there are many reactions.