key: cord-0792381-r0a6a1jw authors: Islam, M. T.; Alam, A. S. M. R. U.; Sakib, N.; Hasan, M. S.; Chakrovarty, T.; Tawyabur, M.; Islam, O. K.; Al-Emran, H. M.; Jahid, I. K.; Hossain, M. A. title: A rapid and cost-effective multiplex ARMS-PCR method for the simultaneous genotyping of the circulating SARS-CoV-2 phylogenetic clades date: 2020-10-13 journal: nan DOI: 10.1101/2020.10.08.20209692 sha: e0b5f8ca2160f5e86a56c447735d268bf252ea82 doc_id: 792381 cord_uid: r0a6a1jw Tracing the globally circulating SARS-CoV-2 mutants is essential for the outbreak alerts and far-reaching epidemiological surveillance. The available technique to identify the phylogenetic clades through high-throughput sequencing is costly, time-consuming, and labor-intensive that hinders the viral genotyping in low-income countries. Here, we propose a rapid, simple and cost-effective amplification-refractory mutation system (ARMS)-based multiplex reverse-transcriptase PCR assay to identify six distinct phylogenetic clades: S, L, V, G, GH, and GR. This approach is applied on 24 COVID-19 positive samples as confirmed by CDC approved real-time PCR assay for SARS-CoV-2. Our multiplex PCR is designed in a mutually exclusive way to identify V-S and G-GH-GR clade variants separately. The pentaplex assay included all five variants and the quadruplex comprised of the triplex variants alongside either V or S clade mutations that created two separate subsets. The procedure was optimized in the primer concentration (0.2-0.6 M) and annealing temperature (56-60{degrees}C) of PCR using 3-5 ng/l cDNA template synthesized upon random- and oligo(dT)-primer based reverse transcription. The different primer concentration for the triplex and quadruplex adjusted to different strengths ensured an even amplification with a maximum resolution of all targeted amplicons. The targeted Sanger sequencing further confirmed the presence of the clade-featured mutations with another set of our designed primers. This multiplex ARMS-PCR assay is sample, cost-effective, and convenient that can successfully discriminate the circulating phylogenetic clades of SARS-CoV-2. SARS-CoV-2 has spread across 188 countries/regions within the first six months of COVID-66 19 pandemic infecting more than 354 million people (Dong, Du, & Gardner, 2020) . This highly 67 infectious virus poses a single-stranded positive-sense RNA genome of nearly 30 kbp 68 (Mousavizadeh & Ghasemi, 2020) . Both synonymous and non-synonymous mutations were 69 identified in the genomic region that code for non-structural proteins (NSP1-16), structural 70 proteins (spike, membrane, envelope, and nucleocapsid proteins), and/or seven other accessory 71 proteins (ORF3a, ORF6, ORF7a, ORF7b, ORF8a, ORF8b, ORF8, and ORF10) (M. R. Islam 72 et al., 2020; Kamitani, 2020; Liu, Fung, Chong, Shukla, & Hilgenfeld, 2014; Ou et al., 2020) . 73 Researchers have demonstrated that the predominant mutations may attribute to virulence 74 (Alam, Islam, Hasan, et al., 2020; Rahman et al., 2020; Q. Wang et al., 2020) . The virus has February 2020 and GR mutant are now the leading type that cause more than one-third of 92 infection globally (Mercatelli & Giorgi, 2020) . Therefore, it is indispensable to identify the 93 circulating clades in a specific region. Besides, several reports speculated the occurrence of 94 SARS-CoV-2 reinfection by phylogenetically different strains that belongs to separate clades 95 (Li et al., 2020; To et al., 2020) . The dominance of a particular viral clade over others might 96 determine the virulence, disease severity, and infection dynamics (Alam et al., 2020) . However, genome center lab. A total of 24 randomly selected SARS CoV-2 positive samples were tested 126 for the analysis (supplementary Table s1 ). A representative SARS CoV-2 negative sample 127 (of 5 of the negative samples randomly selected) was used as a negative control in this study 128 (supplementary Table s2 ). cDNA was prepared for each selected sample using the GoScript™ 5 Reverse Transcription System (Promega, USA) following the manufacturer's protocol. In 130 brief, primer/ RNA mix was prepared by mixing 10µl of extracted RNA with 1µl of Random 131 primer and 1µl of Oligo(dT)15 primer (total volume 12µl). Then the mixture was heated at 70°C 132 for 5 minutes, followed by immediate chilling on ice for 5 minutes and a quick spin. The 133 mixture for reverse transcription reaction was prepared by making a cocktail of the components 134 from GoScript™ Reverse Transcription System in a sterile 1.5ml micro centrifuge tube keeping 135 on ice. The final reaction mix was 40μl for each cDNA synthesis reaction to be performed. 137 A set of 15 primers ( Table 1) was designed based on the ARMS for differentiating six major 138 clades of SARS-CoV-2: S, L, V, G, GH, and GR. We designated here the L clade strains as the 139 wild type and others as mutants. For each clade apart from L, we selected a single representative for ARMS technique, this specificity was directed towards the 3′-end of the annealed primer-147 template (Fig. 1) . The forward or reverse type-specific primers were paired with counterpart 148 reverse or forward primer. The amplicons were simultaneously distinguished by their 149 molecular weight (bp) in multiplex PCR in different combinations. The positive amplification 150 of wild type targeting primers was determined as the L type. The other types were determined 151 based on the co-evolving mutation at respective sites. The primer sets were designed using Primer3Plus (Untergasser et al., 2012) and Primer-153 BLAST (Ye et al., 2012) with the following stringent parameters and standard PCR conditions: 154 avoiding hypothetical primer dimer (self or hetero) formation with less than -9 Kcal/mol, sized 155 18-22 nucleotide in length, Tm of (58-60)°C, (40-60)% GC content, G/C within the last five 156 bases, no repeat of four or more of any base, amplicon size ranging from 200 to 600 bp, and A gradient PCR (SimpliAmp Thermal Cycler, Applied Biosystems, USA) was performed for 163 each of the variant separately with freshly prepared cDNA template to standardize the 164 annealing temperature. Couple of distinct tubes was prepared for each of the variant using the 165 respective primer pairs to differentiate between the wild type and the mutant. The PCR was 166 carried out in 10µl reaction volume containing 3-5 ng/µl DNA, 5µl master mixture (GoTaq® 167 G2 Green Master; Promega, USA), 0.2µM of each forward and reverse primer and 2.8 µl 168 nuclease free water. The thermocycling conditions were as follows: initial denaturation at 95°C 169 for 1 min followed by 30 cycles at 95°C for 30s, annealing at a range of 55-65°C for 30s and 170 72°C for 30s followed by a final extension at 72°C for 5min. The PCR products were 171 electrophoresed on a 1% (w/v) agarose gel stained with ethidium bromide (UltraPure™ 172 Ethidium Bromide, 10 mg/mL; Thermo Fisher, USA) and visualized using a gel documentation 173 system (Bio-Rad, USA). 175 Four sets (duplex, triplex, quadruplex and pentaplex) of multiple variant-specific reactions 176 were arranged for simultaneous detection of a clade. A duplex PCR was performed by using a reproducibility (described in more detail below) was checked over the rest of the samples. 192 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . The multiplex PCR assays were performed over all the 24 positive samples. To validate the 193 reliability of the assays, another five pairs of primer set ( Table 2) Prediction of Primer dimer formation and RNA secondary structure 212 We carried out RNA secondary structure prediction of the ORF3a or NS3 RNA using the Mfold 213 web server (Zuker, 2003) . The full NS3 sequence was extracted from the SARS-CoV-2 214 reference sequence of NCBI GenBank. The default parameters were used in generating 222 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. respectively) (Fig.2 IA-IE) . Hence, the single-variant specific PCRs were able to identify the 228 SARS-CoV-2 positive sample containing GR-clade of the virus. Optimization of multiplex PCR assays 230 The singleplex PCRs showed successful annealing at 57°C, however, temperatures for the 231 duplex, triplex and quadruplex assays were needed to be further optimized to 60°C, 57°C and described before. Besides, the triplex PCR also amplified the products as expected (i.e., 208bp, 242 279bp and 387bp). One of the subsets of quadruplex that contained the variants of triplex plus 243 28144C T>C (p.L84S) was able to distinguish the desired bands individually. However, 244 quadruplex (that had 26144 G>T (p.G251V)) and pentaplex arrangement could not 245 discriminate the bands between wild types and mutants (supplementary Fig.s1 ). 247 All the 24 positive samples confirmed the test reproducibility of the assays; four of them 248 excluding the one used before for multiplex assays were taken as representative to display the 249 reproducibility in this article (Fig.2 IIIA-IIIC) . In this study, only GR clade was found in all 250 positive samples tested. The homology of the nucleotide sequences for the PCR products 251 showed more than 99% identity with the respective positions of the clades that validated the 252 assays (supplementary Fig.s2) . Accession IDs to the submitted sequences for one positive 253 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209692 doi: medRxiv preprint 9 sample as an archetype are available in GISAID EpiFlu™ database (EPI_ISL_548260, 254 EPI_ISL_561630, EPI_ISL_561375, EPI_ISL_561376 and EPI_ISL_561377). This study proposes a simple and exclusive ARMS-based SNP-discriminating method using 257 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 Kcal/mol, but can make longer products ~40 bp. In case of reverse primers that target mutation, 281 only NS3_26144_wR would form a self-dimer with high free energy (-12.9 Kcal/mol). These perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. run (the cost includes import Tax and VAT etc. for Bangladesh) that is much less than targeted 302 and whole-genome based NGS methods in identifying the clades. The cost will be further 303 reduced if an optimized one-step PCR system is used and we are currently working on it to cut 304 the overall cost down to less than $2. Thus, our method can overcome a serious limitation to 305 effectively identify viral clades with a prospective broader application. The requirement of 306 technical skill would also be low for this assay wherein the training of personnel is a minimal 307 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 309 same time. The false-negative result for the absence of a template can also be determined in a 310 facile manner (Edwards & Gibbs, 1994) . In general, mutating the primer at its 3'prime end personnel to analyze NGS data is also a reason to prefer alternative approach other than NGS 318 technology in low-income countries. Therefore, the ARMS technology with the conventional 319 multiplex PCR methods in identifying the clades would be more applicable in low and 320 minimum resource settings. 321 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. Our assay can enhance the identification of genotypic variants of SARS-CoV-2 worldwide, 323 especially in low-resource settings where NGS and Sanger sequencing techniques are difficult 324 to reach out. This rapid barcoding method may assist to reveal disease epidemiology, patient 325 management and protein-based drug designing, and also contribute to modify future national 326 policy, and vaccine development. A more cost-effective one-step procedure based on modified 327 tetra ARMS assay (T-ARMS) is under development by our group that will considerably reduce 328 the labor and cost further. Acknowledgments 330 We acknowledge GISAID for sharing the sequence data and IDT for giving the opportunity to 331 use the tools for validating primers in silico. We also acknowledge Ministry of Health and 332 family Welfare, Bangladesh for giving us the permission for SARS CoV-2 diagnosis. The authors declare no completing interest to this work. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10. 1101 /2020 This study was approved by the ethical review committee of Jashore University of Science and perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020 . . https://doi.org/10.1101 /2020 perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209692 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209692 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209692 doi: medRxiv preprint Table 531 1. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. Table 1 . perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209692 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . GC' indicates the Genome Center identification number generated at the Genome Center 559 of Jashore University of Science and Technology for COVID-19 suspected patients