RT-PCR tests: What it is and how it is done? 

According to microbiologists, RT-PCR revolutionized the way clinical microbiology laboratories diagnose many human microbial infection

Published: 21st May 2020 11:26 PM  |   Last Updated: 21st May 2020 11:26 PM   |  A+A-

Health department staff collecting swabs from police personnel and their family members to detect Coronavirus infection through RT-PCR method at Coimbatore Railway Station on Monday. (Photo | U Rakesh Kumar/EPS)

By Express News Service

THIRUVANANTHAPURAM:  For the detection of Covid-19, Real-Time-Polymerase Chain Reaction Test, most commonly-known as RT-PCR test is being widely used. But this RT-PCR is not limited to detection of the SARS-CoV-2. According to microbiologists it revolutionized the way clinical microbiology laboratories diagnose many human microbial infection. 

According to the American Society for Microbiology, the combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases? 

How RT-PCR is used for detecting Covid-19? 

The causative agent for Covid19 is the SARS-CoV-2 virus. It is an RNA virus, that means it infiltrates a healthy cell to multiply and survive. Thus the RT-PCR test is for the identification of SARS-CoV-2 RNA. In this, the RNA is converted to DNA through a process called 'reverse transcription' for detecting viruses.

The SARS-CoV-2 RNA is generally detectable in respiratory specimens during the acute phase of infection.  For that upper and lower respiratory specimens (such as nasal, nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage, and nasopharyngeal wash/aspirate or nasal aspirate) are collected. This sample is treated with several chemical solutions that remove substances, such as proteins and fats, and extracts only the RNA present in the sample. 

How results are arrived? 

As per an international document standard real-time RT-PCR setup usually goes through 35 cycles, which means that by the end of the process, around 35 billion new copies of the sections of viral DNA are created from each strand of the virus present in the sample. 

As new copies of the viral DNA sections are built, the marker labels attach to the DNA strands and then release a fluorescent dye, which is measured by the machine's computer and presented in real-time on the screen. The computer tracks the amount of fluorescence in the sample after each cycle. When the amount goes over a certain level of fluorescence, this confirms that the virus is present. 

Scientists also monitor how many cycles it takes to reach this level in order to estimate the severity of the infection: the fewer the cycles, the more severe the viral infection is.

Does the results are hundred percent accurate? 

No. The state itself had incidents of false positives and false negatives. Positive results are indicative of the presence of SARS-CoV-2 RNA. But it does not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. Also, negative results do not preclude the infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.

Who could carry out the test? 

Only trained clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures could carry out the RT-PCR Tests. In Kerala, 19 labs conduct RT-PCR tests. This includes 14 government labs and five private labs.     

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