The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β 2 -microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected ≤ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.
【저자키워드】 COVID-19, SARS-CoV-2, molecular diagnostics, genetic variants, real-time TaqMan reverse transcription PCR assays, locked nucleic acid (LNA), 【초록키워드】 Efficacy, coronavirus, pandemic, Mutation, spike, variant, SARS-CoV-2 variant, Infection, virus, Spread, early diagnosis, amplification, nucleic acid, rRT-PCR, Internal control, Patient, Respiratory pathogens, envelope, NL63, mutant, reverse transcription PCR, Quantitative, OC43, False-positive, 229E, nucleotide, SARS-CoV-2 viral RNA, viral strain, acute respiratory syndrome, sequence, wild-type, circulating, selective, positive, contagious, TaqMan, coding sequence, transmission of SARS-CoV-2, probe, dominant, Genes, approach, populations, Prevent, RNA transcripts, human coronavirus, clinical sample, Result, identify, evaluated, required, nine, linear, exhibited, amplify, quantified, the Spike, B2M, circulating variant, primer-probe set, 【제목키워드】 coronavirus, variant, detection, reverse transcription-PCR, Quantitative, acute respiratory syndrome,