How are novel corona-virus COVID-19 tests done?

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Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a technique of making numerous copies of specific segment of DNA/RNA accurately and quickly. It was developed by K. B. Mullis, an American Biochemist, in 1983. This is based on the natural process a cells uses to create new DNA/RNA (component of gene and its expression). In this method few biological ingredients are added, resulting in vitro chemical reaction that permits synthesis of essentially limitless quantity of the targeted nucleic acid sequence.

A PCR cycle consists of three steps: denaturation, annealing and extension. At the end of each cycle, PCR products are doubles. Hence after, n-PCR cycles, the target sequence can be amplified exponentially with 2. Ideally after 20 cycles of PCR, a million fold amplifications are obtained.

In context of COVID-19

Out of many types of PCR, “Reverse-transcription PCR” has ability to amplify ribonucleic acid (RNA) targets. The reverse transcription method creates complementary DNA from RNA targets and then these complementary DNA is amplified. This method is used in detection of Hepatitis-C virus RNA, for quantification of HIV and recently for early and accurate diagnosis of COVID-19.

Sample and processing

PCR
Pic: Cytogeneticist Dr. Maharjan with PCR

In order to test COVID-19, samples should be obtained from oropharyngeal (preferred) if not nasal mid turbinate or sputum. However, bronchoalveolar lavage, although highly sensitive, is less commonly used due to poor patient compliance. Specimens should be collected as soon as possible once decision is made to pursue COVID-19 test and sample should be stored at 2-8 degree Celsius (Ice pack can be used). These samples should be processed in Biological safety level-2 labs, which should be featured by appropriate PPE, Biological safety cabinet, self-closing and lockable door, sink eyewash station, autoclave and biohazard warning sign. However, according to the Center for Disease Control, USA strongly recommend laboratory employee working with suspected Corona virus samples, should operate with HEPA(high-efficiency particulate absorbing) filtered powered air-purifying respirator and a facility with negative relative air pressure with respect to surrounding spaces with exhaust air vented directly to outside environment.

For utilization of the PCR test, in disease diagnosis, nuclei acid sequence (template) of target region which should be unique in the disease causing pathogen is must. Other ingredient includes sequence specific primers, thermo-stable polymerase, nucleotides, buffer etc.

PCR contamination, once established is very difficult to get rid of; hence best approach is to prevent from happening in the first instance.
Good training of laboratory staffs is essential, who ultimately determine how the laboratory function. In the current scenario, one false positive or negative result can cause lots of suffering, precision and accuracy of the test should be of utmost importance.

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Author: @DrShanroc

Dr. Shankar Bastakoty B.P. Koirala Memorial Cancer Hospital