Different countries used different protocols for RT-PCR diagnostic testing for SARS CoV-2. Some countries target the N region gene of SARS CoV-2. This is because, among other reasons, it is a highly conserved region in coronaviruses genome and to deliver consistent results. so basically we need to target specific genes and perform RT-PCR test.
All sequence of SARS CoV-2 first provided by china in december 2019. https://www.ncbi.nlm.nih.gov/nuccore/NC_045512
METHOD of RT PCR
First take sample through nasopharyngeal or oropharyngeal swap. Once swap is take then immediately transfer into viral transport medium(to protect viral genome). Then extract RNA using buffer solution.Conventionally in PCR DNA is used for amplification process so first convert RNA into DNA by using reverse transcriptase.
Then perform PCR followed by three steps
- DENATURATION:- separate the DNA double helix
- ANNEALING:- Primers binds the specific region(N-region) and starts polymerisation
- EXTENSION:- New strands of DNA are made using original strand are templates DNA polymerase enzyme joins free DNA nucleotide together.
PCR is cycle process repeated many times in 2-3 hours generate billion of copies.
To get result we need to know PCR annealing step in detail
Primers are small fragments of ssDNA designed to bind specifically to a single region in the genome to allow for precise amplification of the diagnostic target area. Primers bind to the target area and give off a flurescent signal as amplification of the area increases. This flurescent signal is then read by the quantitative real PCR machine where the reaction is occuring to give a result positive or negative.
In india COVID-19 test conducted as per kits approved by ICMR(indain council of medical research)