Abstract
COVID-19 is caused by SARS-CoV-2, several virulent variants of which have emerged since 2019. More than 529 million people have been infected, and at least 6 million have died. Our aim was to develop a fast, accurate, low-cost method for detecting and identifying newly emerging variants of concern (VOCs) that could pose a global threat. The 341-bp DNA sequence of a specific region of the SARS-CoV-2’s spike protein was amplified by a one-step PCR on RNA samples from 46 patients. The product was sequenced using next-generation sequencing (NGS). DNA sequences from seven genomes, the original Wuhan isolate and six different representative variants obtained from the GISAID website, were used as references. Complete whole-genome sequences from local isolates were also obtained from the GISAID website, and their RNA was used for comparison. We used an amplicon-based NGS method (termed VOC-NGS) for genotyping and successfully identified all 46 samples. Fifteen (32.6%) were like the original isolate. Twenty-seven were VOCs: nine (19.5%) Alpha, eight (19%) Delta, six (14%) Beta, and four (8.7%) Omicron. Two were variants of interest (VOI): one (2%) Kappa and one (2%) Zeta. Two samples were mixtures of two variants, one of Alpha and Beta and one of Alpha and Delta. The Spearman correlation between whole-genome sequencing (WGS) and VOC-NGS was significant ( P < 0.001) with perfect agreement (Kappa = 0.916) for 36/38 (94.7%) samples with VOC-NGS detecting all the known VOCs. Genotyping by VOC-NGS enables rapid screening of high-throughput clinical samples that includes the identification of VOCs and mixtures of variants, at lower cost than WGS. IMPORTANCE The manuscript described SARS-Cov-2 genotyping by VOC-NGS, which presents an ideal balance of accuracy, rapidity, and cost for detecting and globally tracking VOCs and some VOI of SARS-CoV-2. A large number of clinical samples can be tested together. Rapid introduction of new mutations at a specific site of the spike protein necessitates efficient strain detection and identification to enable choice of treatment and the application of vaccination, as well as planning public health policy.
Keywords: NGS; PCR; SARS-CoV-2; WGS; mutations; pandemic; variants of concern (VOC); variants of interest (VOI).
【저자키워드】 SARS-CoV-2, pandemic, NGS, mutations, PCR, variants of concern (VOC), WGS, Variants of interest (VOI)., 【초록키워드】 COVID-19, Treatment, whole-genome sequencing, public health, vaccination, Mutation, VoC, variant, variants of concern, Delta, Local, omicron, variants, Spike protein, RNA, Accuracy, Next-generation sequencing, VOCs, Rapid, variants of interest, Beta, genomes, correlation, genotyping, GISAID, patients, website, manuscript, DNA sequence, virulent, Whole-genome sequence, Spearman, isolate, Seven, clinical sample, tested, amplified, described, was used, develop, caused, sequenced, died, include, nine, eight, were used, the spike protein, RNA sample, one-step PCR, the SARS-CoV-2, Wuhan isolate, 【제목키워드】 concern, Amplicon, identification,