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Reverse Transcription PCR technique to Detect COVID 19


Corona virus is an infectious disease and is caused by severe acute respiratory syndrome corona virus 2 (SARS COV-2). The symptoms include high fever, cough and shortness of breath. The other symptoms include body, abdominal pain, sore throat and diarrhea. The disease first appeared in China and is spreading globally creating a pandemic situation.

Here is the link to the fasta sequence of Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome


COVID 19 RT PCR is a real time reverse transcription polymerase chain reaction used for detection of nucleic acid from SARS-CoV2 obtained from the respiratory specimens (sputum and swabs) of the suspected patients. Positive results are the indicative for the presence of SARS CoV2 RNA. Laboratory Corporation of America uses Roche MagNA pure 96 instrument and Applied Biosystems QuantStudio 7 Flex instrument version 1.3.
The test uses 3 primers and probe sets to detect three regions in corona virus nucleocapsid gene, one primer and probe set to detect human RNAse in clinical sample. The RNA is reverse transcribed into CDNA and can be amplified using QuantStudio 7 flex instrument. This procedure uses a modified thermal cycler with UV Detector and Charged couple device (CCD) camera. The procedure measures the amount of amplicon produced in each cycle. Probe is an oligonucleotide consists of reporter and quencher dye. When the reporter is excited by the light it transfers the energy to the quencher and prevents reporter dye to emit light. The cleavage of the probe takes place during the extension of the PCR reaction. When the light excites the reporter dye, the reporter dye can now emit light since the quencher dye is far to block the emission.
Four different controls should be used along with COVID 19 RT PCR.
• Negative control (no template) should be used to eliminate sample and reagent contamination on the assay run.
• A positive control (COVID 19 _N_P) is used to check whether the run is working and is used on every run plates at a concentration of 50 copies per microliters.
• An internal control is needed to verify nucleic acid is present in every sample.
• Negative extraction control is also used in the procedure to monitor for any cross contamination during extraction process.

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