Discussion
This study proposes a simple and exclusive ARMS-based SNP-discriminating
method using conventional PCR to establish multiplex-assays in detecting
SARS-CoV-2 mutation clades. This concept was adopted from the other
studies applied to identify the genetic profile of respiratory or
gastrointestinal coronaviruses of pigs, human cancer risk related SNPs,
virus that causes systemic infection in canines, the resistance profile
of a bacteria etc. (Chulakasian et al., 2010; Lai, Welter, & Welter,
1995; Shi et al., 2013; C. Zhang et al., 2013). This study designed
point-mutation specific primers to detect the six different SARS-CoV-2
clades as described by GISAID. The clade-based discrimination during
COVID-19 pandemic was exceedingly important because the prevalence of
SARS-CoV-2 clades were varied by regions and times, and were closely
related to variable death-case ratio (Alam et al., 2020; Toyoshima et al
2020, nature). G clade variant was dominant in Europe (Korber et al.,
2020) and USA (Brufsky, 2020) on the beginning of the pandemic that
caused high mortality in USA. This mutation variant was gradually
circulating in Southeast Asia (Alam, Islam, Hasan, et al., 2020; O. K.
Islam et al., 2020) and Oceania (Mercatelli & Giorgi, 2020). Among the
two derivative of G clade (GR and GH) that emerged at the end of
February 2020, GR mutant are now the leading type that cause more than
one-third of infection globally (Mercatelli & Giorgi, 2020).
In this study, we attempted to validate two sets of multiplex PCR
covering G, GH and GR in the first set, and V and S in the second set.
Based on the available data of clade prevalence we propose to run the
first set of multiplex PCR in the beginning that can confirm the most
three prevalent clades (Alam, Islam, Hasan, et al., 2020). Our attempt
for pentaplex and/or the quadruplex (that included the SNP variant 26144
G>T (p.G251V)) was unsuccessful, where the template
regarding the variant 26144 G>T (p.G251V) did not amplify,
possibly due to primer-dimer formation with higher thermodynamic
stability than other variant-specific primer sets. The forward primer
could bind to the N_28882_mR primer with a G value of <-7
Kcal/mol, but can make longer products ~40 bp. In case
of reverse primers that target mutation, only NS3_26144_wR would form
a self-dimer with high free energy (-12.9 Kcal/mol). These homo- and
hetero-dimer formation would make more primer duplex and might reduce
the chance of effective pentaplex PCR. Another possibility could be that
the NS3 binding region of the primers (205-222 and 752-772) has resided
in a stable stem site hindered the effective annealing. The RNA
structure showed the complex stem-loop region and open sites as well.
Our targeted primer binding sites for the variant 26144 G>T
(p.G251V) were within the stem-loop region whereas the primer annealing
sites for 25563 G>T (p.Q57H) variant reside within the open
region of the template. Here we assume, the complex structural
configuration of NS3 may block the PCR reaction during a competitive
multiplexing (supplementary Fig.s3 ). A longer RNA denaturation
step during cDNA synthesis and more stringent cycle denaturation of cDNA
template might solve this issue. However, it could damage
low-concentrated, sample-extracted viral RNA and inhibit amplification
of other clade-specific templates by affecting overall optimized
multiplex condition.
The advantage of our ARMS-based multiplex assays is rapid. The
turnaround time for our designed assay would range from approximately
3-4 hours for 96 samples. The NGS and Sanger methods, on the other hand,
had a turnaround time of more than 24 hours and 10-12 hours,
respectively (Tsuchihashi & Dracopoli, 2002; J. Zhang et al., 2020). An
ARMS based multiplex PCR assay similar to the current study would render
a more convenient way to detect clade specific mutation (SNPs) due to
the process being faster and cost effective (Ahlawat, Sharma, Maitra,
Roy, & Tantia, 2014). The cost of the assay for a single reaction was
$7 per run (the cost includes import Tax and VAT etc. for Bangladesh)
that is much less than targeted and whole-genome based NGS methods in
identifying the clades. The cost will be further reduced if an optimized
one-step PCR system is used and we are currently working on it to cut
the overall cost down to less than $2. Thus, our method can overcome a
serious limitation to effectively identify viral clades with a
prospective broader application. The requirement of technical skill
would also be low for this assay wherein the training of personnel is a
minimal requirement and interpretation of results is generic (Syrmis et
al., 2004; L. Wang et al., 2011).
Besides, the presence of the template as well as their quantity and
quality are determined at the same time. The false-negative result for
the absence of a template can also be determined in a facile manner
(Edwards & Gibbs, 1994). In general, mutating the primer at its 3’prime
end makes it refractory to the ‘wild type template’ whereas the absence
of mutation in the primer is retractable to the ‘mutant template’
amending a reliable technique over sequencing (Chulakasian et al.,
2010). On the other hand, next generation sequencing technology such as
whole genome sequencing (WGS) or metagenomics approach can generate
millions of high-throughput data that enabled researchers to unroll new
dimensions in the field of genome sequencing applications (El-Metwally,
Hamza, Zakaria, & Helmy, 2013). The lack of technical personnel to
analyze NGS data is also a reason to prefer alternative approach other
than NGS technology in low-income countries. Therefore, the ARMS
technology with the conventional multiplex PCR methods in identifying
the clades would be more applicable in low and minimum resource
settings.