Page 27 - Dream May 2020 English

COVID-19 SPECIAL
TESTING
The world is in the midst of a pandemic of coronavirus disease 2019, also known as COVID-19. The causative agent responsible for COVID-19 is a virus called severe acute respiratory syndrome coronavirus 2, or SARS-CoV-2. Initially known as the novel coronavirus, the new virus has been given the name SARS-CoV-2 by the International Committee of Taxonomy of Viruses (ICTV).
Coronaviruses have ribonucleic acid (RNA) as the genetic material. The spherical particles have spike-shaped proteins protruding from their surface. These spikes latch onto human cells, then undergo a structural change that allows the viral membrane to fuse with the cell membrane. The viral genes can then enter the host cell to be copied, producing more viruses. This type of virus can replicate their RNA genomes with the help of RNA dependent RNA polymerases.
In the urgent situation for combating this disease a viral genome sequence was released via the community online resource virological.org on 10 January 2020. After this, four other genomes were deposited on 12 January 2020 in the viral sequence database curated by the Global Initiative on Sharing All Influenza Data (GISAID).
For tackling the spread of SARS-CoV-2 infection, a reliable laboratory diagnosis is most important for the identification of infected persons and their proper quarantine and treatment. As in cases of acute respiratory infection, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is routinely used to detect causative viruses in respiratory secretions. Other tests may include serological assays and viral culture. But as the RT-PCR is rapid, specific and reliable compared to other tests, it is widely accepted for the diagnosis of COVID-19.
As per the WHO, the decision to test should be based on clinical and epidemiological factors. PCR testing of suspected individuals with symptoms or without one can be considered for diagnosis of infection of COVID-19. PCR is uniquely suitable for the identification of a small quantity of genome of the virus. The method leads to the amplification of the desired gene of the virus and then its identification.
Historically, the invention of the PCR is credited to American biochemist Kary Mullis in 1983. Its principle is based on the DNA polymerase action, which is in vitro replication of specific DNA sequences. This method can produce tens of billions of copies of a particular DNA fragment (the sequence of interest, DNA of interest, or target DNA) from a DNA extract (DNA template). PCR can be used to obtain multiple copies of DNA. The matrix DNA can be a genomic DNA or complementary DNA (cDNA) obtained by RT-PCR from a messenger RNA extract (poly-A RNA) or even mitochondrial DNA.
PCR is a technique for obtaining large amounts of a specific DNA sequence from a DNA sample. This amplification is based on the replication of a double-stranded DNA template. The
PCR has phases: a denaturation phase, a hybridisation phase with primers, and an elongation phase. The products of each synthesis step serve as a template for the next steps; thus, exponential amplification is achieved.
The denaturation
In this step, double-stranded DNA is separated into single- stranded DNA by raising the temperature to 90-98°C.
Hybridisation or annealing
In the second step, which is termed as hybridisation or annealing phase, the mixture is cooled down to 40-60°C which permits complementary sequences of single-stranded DNA or RNA to pair by hydrogen bonds to form a double-stranded polynucleotide.
Elongation
In the third step, temperature is again raised to 70-75°C, which is optimum for action of an enzyme called Taq polymerase to start the attachment of nucleotides on primer as per the complementary sequences in the original DNA. Taq polymerases are named after their bacterium source Thermusaquaticus.
Thus, the new DNA strand gets elongated. After completion of these steps two double-stranded DNAs are produced. Then, again, step 1 begins for getting 4 double stranded DNAs and so, multiplication of DNA proceeds after each cycle.
RT-PCR is sometimes misunderstood as a real-time polymerase chain reaction. However, they are separate and distinct techniques. Application of RT-PCR for the qualitatively detection of gene expression involves creation of complementary DNA (cDNA) transcripts from RNA. As the SARS-CoV2 virus is an RNA virus, it can be detected by using RT-PCR. In case of RT-PCR, the enzyme reverse transcriptase first produces copy of DNA from RNA and then the steps of PCR begin.
Serological testing
Serological surveys can be performed in addition to the investigation of an ongoing epidemic where nucleic acid amplification tests (NAAT), such as RT-PCR assays are negative and there is a strong epidemiological link to COVID-19 infection. The serum samples may support diagnosis if validated serology tests are available. Serum samples can be stored for these purposes. The possible problem with serological tests may include the cross-reactivity of other coronaviruses. Commercial and non-commercial serological tests for SARS- CoV2 are currently under development.
The author is Scientist ‘C’ in Vigyan Prasar. Email: sachin@vigyanprasar.gov.in
Technologies for the diagnosis of COVID-19 Sachin C. Narwadiya
11 dream2047/may2020