Introduction
SARS-CoV-2 has spread across 188 countries/regions within the first six
months of COVID-19 pandemic infecting more than 354 million people
(Dong, Du, & Gardner, 2020). This highly infectious virus poses a
single-stranded positive-sense RNA genome of nearly 30 kbp (Mousavizadeh
& Ghasemi, 2020). Both synonymous and non-synonymous mutations were
identified in the genomic region that code for non-structural proteins
(NSP1-16), structural proteins (spike, membrane, envelope, and
nucleocapsid proteins), and/or seven other accessory proteins (ORF3a,
ORF6, ORF7a, ORF7b, ORF8a, ORF8b, ORF8, and ORF10) (M. R. Islam et al.,
2020; Kamitani, 2020; Liu, Fung, Chong, Shukla, & Hilgenfeld, 2014; Ou
et al., 2020). Researchers have demonstrated that the predominant
mutations may attribute to virulence (Alam, Islam, Hasan, et al., 2020;
Rahman et al., 2020; Q. Wang et al., 2020). The virus has been
classified into six clades namely GH, GR, G, V, L, and S by the global
initiative on sharing all influenza data (GISAID) (Shu & McCauley,
2017) by the clustered, co-evolving, and clade-featured point mutations.
The mutations at position C241T along with C3037T, C14408T
(RdRp:p.P323L), and A23403G (S:p.D614G) was referred as G clade.
Additional mutation to the G clade at N protein:p.RG203-204KR
(GGG28881-28883AAC) and ORF3a:p.Q57H (G25563T) refers to GR and GH
clade, respectively. The V clade was classified by co-evolving mutations
at G11083T (NSP6:p.L37F) and G26144T (ORF3a:p.G251V) where S clade
strains contain C8782T and T28144C (NS8:p.L84S) variations,
respectively. The L clade strains are the original or wild version for
the featured mutations of five clades (Mercatelli & Giorgi, 2020).
Previous studies showed the prevalence of phylogenetic clades were
different by regions and times and were closely related to variable
death-case ratio (Alam et al., 2020; Toyoshima et al 2020). On the eve
of pandemic, G clade variant was predominant in Europe (Korber et al.,
2020) and USA (Brufsky, 2020) where this clade caused high mortality in
USA. This variant has gradually been spreading in Southeast Asia (Alam
et al., 2020; Islam et al., 2020) and Oceania (Mercatelli & Giorgi,
2020). On the contrary, GR and GH clades emerged at the end of February
2020 and GR mutant are now the leading type that cause more than
one-third of infection globally (Mercatelli & Giorgi, 2020). Therefore,
it is indispensable to identify the circulating clades in a specific
region. Besides, several reports speculated the occurrence of SARS-CoV-2
reinfection by phylogenetically different strains that belongs to
separate clades (Li et al., 2020; To et al., 2020). The dominance of a
particular viral clade over others might determine the virulence,
disease severity, and infection dynamics (Alam et al., 2020). However,
the implications of different clades on effective drug and vaccine
development is yet to be clearly elucidated (Chellapandi & Saranya,
2020).
The identification of phylogenetic clades requires the identification of
specific mutations into viral genome. This identification is performed
by the whole genome sequence through NGS technique that has now scaled
up the deposited sequences number in GISAID to 139,000 as of October 6,
2020. Another high-throughput NGS alternative is based on clade-based
genetic barcoding that targets PCR amplicons encompassing the featured
mutation as described by Guan et al. (2020). However, this state of art
technique has limited access to most laboratories in low-income
countries. A short-throughput and small-scale genotyping would be the
Sanger based targeted sequencing approach (Alam, Islam, Rahman, Islam,
& Hossain, 2020), but this is labor intensive, time-consuming,
inconvenient, and difficult to perform at low cost. Therefore, we have
hardly observed the worldwide distribution of circulating clades in many
countries, like Afghanistan, Maldives, Iraq, Syria, Yemen, Ethiopia,
Sudan, Zimbabwe, Bolivia, Paraguay, and Chile, most probably due to the
lack of sequencing facilities and appropriate technical personnel to
perform this state-of-the-art technique. PCR-based point mutation
discriminating technique, which is also known as the amplified
refractory mutation system (ARMS), has been proven to be useful in
identifying subtypes or clades of other respiratory viruses previously
(Brister, Barnum, Reedy, Chambers, & Pusterla, 2019; Lee, Kim, Shin, &
Song, 2016; W. Wang et al., 2009). In this study, we aimed to develop
and validate an ARMS-based novel multiplex-PCR to identify the
clade-specific point mutations of the circulating SARS-CoV-2 clades.