RT PCR is considered the “gold standard” for testing the presence of the novel SARS CoV2 virus in a swab specimen taken from a patient suspected to have COVID-19. Testing involves the following steps in the workflow: (1) Sample reception, encoding and inspection; (2) Inactivation and Ribonucleic acid (RNA) Extraction; (3) Reverse Transcription – Polymerase Chain Reaction (RT PCR); (4) Results Generation, Interpretation, and Validation.

During sample reception, the samples that arrive from different hospitals and community quarantine centers are encoded into the laboratory database and they are given a unique specimen identification number. The samples are cross-checked with the patient’s name and specimen ID and are then inspected for sample leakage under a biosafety level 2 cabinet with the staff in full personal protective equipment (PPE) since the samples contain live viruses. If there is leakage, these are discarded as biohazard waste; and are then reported back to hospital of source. The next step is inactivation of the virus either by heat or chemical means; this process renders the virus non-infective. Heat inactivation involves incubation at 65 degrees Centigrade using a heat block for about 10 minutes, immediately followed by RNA extraction. Chemical inactivation, on the other hand, uses chemicals that breakdown the viral components such as a lysis buffer included in RNA extraction kits.

During the extraction step, RNA is isolated and purified, a vital step to be able to perform a good PCR. RNA being a single-stranded molecule is highly unstable, it needs to be converted (or reverse transcribed) to a more stable form called complementary deoxyribonucleic acid or cDNA. In this more stable form, the DNA can be made into multiple copies using the PCR machine.

There are different PCR kits available for use, each of these kits detect different parts of the SARS CoV 2 gene that carry directions for making proteins that are needed for the virus to replicate inside the human cell. The primary objective of the COVID-19 test is to identify parts of the viral genome present in the sample. Using a real-time PCR and the use of fluorescent probes, amplification can be monitored in real time and the DNA copies can be quantified. Results, whether the SARS CoV2 was detected or not, are then generated, interpreted, and validated.

The usual turn around time is 48 hours from the time the sample reaches the laboratory. These results are given back to the hospital source and reported to the Regional Epidemiological and Surveillance Unit of the Department of Health. 

Dr. Eva Maria Cutiongco-de la Paz | Published in Healthscape Special COVID-19 Issue No. 11