WHAT IS RT-PCR TEST for detecting corona virus infection? By Dr Sunilkumar Bura
RT-PCR Means Real-Time Polymerase chain reaction. RT-PCR one of the type PCR which the RNA template is first converted into a complementary DNA (cDNA) using reverse transcriptase. The cDNA is then used as a template for exponential amplification using PCR. Enabled sample amplification and eliminated the need for abundant starting material required when using northern blot analysis.
RT-PCR has been used to measure viral load with HIV and may also be used with other RNA viruses such as measles and mumps.
Advantages of real-time PCR test-
1. Amplification and detection occur in a single tube, eliminate post PCR manipulation.
2. An increased dynamic range of detection.
3. Ability to precisely measure the amount of amplicon at each cycle which allows highly accurate quantification of the amount of starting material in samples.
4. Ability to monitor PCR reaction in real-time.
Real-time PCR steps-There are mainly 3 steps involved in real-time PCR Reaction. Reactions generally run for 40 cycles -
1. Denaturation-High temperature incubation is used to melt double standard DNA into single standard DNA and loosen secondary structure in single standard DNA. The highest temperature use for Denaturation is 95 degrees.
2. Annealing-Complementary sequences have an opportunity to hybridize.
3. Extension-At 70-72ºc the activity of the DNA polymerase is optimal, and primer extension occurs at the rates of up to 100 bases per second.
TWO STEP qRT-PCR-Reverse transcriptase PCR (qRT-PCR) starts with the reverse transcription of either RNA or poly (A)+RNA into CDNA.
ONE STEP QRT-PCR-First-strand cDNA synthesis reaction and real-time PCR reaction in the same tube, simplifying reaction setup and reducing the possibility of contamination.
WHAT IS RT-PCR FOR nCOVID-19
The Health Ministry has announced that the real-time reserve transcription-polymerase chain reaction or RT-PCR is the gold standard frontline test for COVID-19.
One is more accurate called RT-PCR which is extracting RNA from the nasal or mouth swab. Because of Genome sequencing technology, we have the sequence of SARS-COV-2 and RNA of the swab is checked whether virus and RNA share the same sequence. Because of proper research and Genome sequence projects going around the world, RT-PCR tests are considered more accurate and reliable.
DISADVANTAGES OF RT-PCR-
RT-PCR Technology is a relatively expensive method. It requires RNA extracting machines, a laboratory and trained technicians. Minimum of 30 samples is needed to make it economically viable.