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).