A rapid and cost‐effective multiplex ARMS‐PCR method for the simultaneous genotyping of the circulating SARS‐CoV‐2 phylogenetic clades
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Tracing the globally circulating severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) phylogenetic clades by high‐throughput sequencing is costly, time‐consuming, and labor‐intensive. We here propose a rapid, simple, and cost‐effective amplification refractory mutation system (ARMS)‐based multiplex reverse‐transcription polymerase chain reaction (PCR) assay to identify six distinct phylogenetic clades: S, L, V, G, GH, and GR. 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 with 0.2–0.6 µM primer concentration, 56–60°C annealing temperature, and 3–5 ng/µl complementary DNA to validate on 24 COVID‐19‐positive samples. Targeted Sanger sequencing further confirmed the presence of the clade‐featured mutations with another set of primers. This multiplex ARMS‐PCR assay is a fast, low‐cost alternative and convenient to discriminate the circulating phylogenetic clades of SARS‐CoV‐2.
Article activity feed
-
SciScore for 10.1101/2020.10.08.20209692: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization A total of 24 randomly selected SARS CoV-2 positive samples were tested for the analysis (supplementary Table s1). Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Experimental Models: Organisms/Strains Sentences Resources NS3_26144_F-NS3_26144_wR (wild type)/NS3_26144_F-NS3_26144_mR (mutant) and NS8_28144_F-NS8_28144_wR (wild type)/ NS8_28144_F-NS8_28144_mR (mutant), respectively while mixing for wild types and the mutants in separate PCR tubes. NS3_26144_F-NS3_26144_wRsuggested: NoneS_23403_wF-S_23403_R (wild type primers)/ S_23403_mF-S_23403_R (mutant primers), … SciScore for 10.1101/2020.10.08.20209692: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization A total of 24 randomly selected SARS CoV-2 positive samples were tested for the analysis (supplementary Table s1). Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Experimental Models: Organisms/Strains Sentences Resources NS3_26144_F-NS3_26144_wR (wild type)/NS3_26144_F-NS3_26144_mR (mutant) and NS8_28144_F-NS8_28144_wR (wild type)/ NS8_28144_F-NS8_28144_mR (mutant), respectively while mixing for wild types and the mutants in separate PCR tubes. NS3_26144_F-NS3_26144_wRsuggested: NoneS_23403_wF-S_23403_R (wild type primers)/ S_23403_mF-S_23403_R (mutant primers), NS3_25563_w1F-NS3_25563_1R (wild type primers)/ NS3_25563_m1F-NS3_25563_1R (mutant), and N_28882_F-N_28882_wR (wild type)/ N_28882_F-N_28882_mR specific to 23403 A>G (p.D614G), 25563 G>T (p.Q57H) and 28882 G>A (p.R203K) SNP variants respectively. S_23403_wF-S_23403_Rsuggested: NoneSoftware and Algorithms Sentences Resources The primer specificity against SARS-CoV-2 and other organisms was checked by the Primer-BLAST. Primer-BLASTsuggested: (Primer-BLAST, RRID:SCR_003095)We performed the in silico PCR with the primers in the UCSC genome browser (https://genome.ucsc.edu/). UCSC genome browsersuggested: (UCSC Genome Browser, RRID:SCR_005780)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study: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.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
-
-
