Polymerase chain reaction
The development of reverse transcriptase polymerase chain reaction (RT-PCR) methods to detect RNA sequences has been a major advance in viral diagnostics (Dembert & Kaiser, 1983). Principally, PCR tests are very specific but less sensitive, therefore, negative test do not rule out infection; positivity does not indicate quantity large enough to be transmitted (WHO, 2003). The highest yield for the detection of SARS-CoVs has been assessed by RT- PCR (Chan et al., 2004); positive RT-PCR results is usually confirmed by repeat testing of the original specimen and through retesting in an independent laboratory using a validated assay (Wadsworth, 2003). This is because PCR is useful at early stage of the disease but can produce many false-negative results, creating dangerous security issues that could aid transmission (Roos, 2003).
Cases of both SARS-CoV and SARS-CoV-2 are routinely confirmed on the basis of detection of RNA sequences that are unique to the viruses by NAAT such as nucleic acid real-time reverse-transcription PCR (rRT-PCR) (Wadsworth, 2003; WHO, 2020). Nucleic acid sequencing can serve as confirmatory test if necessary, especially in areas that is not known with incidence of the disease, hence SARS-like coronaviruses or beta coronaviruses have to be considered before sequencing. In the COVID-19 isolated areas however, a single rRT-PCR can be reliable (WHO, 2020).
Members of the family coronaviridae have between 29,000 to 31,000 nucleotides (CDC, 2003a). The genomic nucleotide length of SARS-CoV is 29,727 and there is similar genomic organisation with that of COVID19. The S and N proteins, small membrane protein (E), membrane protein (M), and several other open-reading frames of unknown function, have been identified with SARS-CoVs (CDC, 2003b). Oligonucleotide primers that are used to detect COVID-19 were selected from the N-region of its genetic region of its nucleocapsid, designed with dual primer/probe sets with additional set for human RNase P (RP) gene as control (CDC, 2020). COVID-19 detector that employ simultaneous use of rRT-PCR and loop-mediated amplification techniques has been produced, which does not require repeated heating and cooling cycles of PCR and the amplified nucleic acid is incubated with a guide RNA (gRNA) molecule that target the viral E and N genes with the help of single strand Deoxyribonucleic acid (ssDNA) that confirm the presence of the target RNA (Sheridan, 2020; Huang et al., 2020).