Abstract
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 β2-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.
Highlights
The human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) started in the Chinese city of Wuhan in late 2019, and spread around the world [1,2,3]
Limits of detection (LoD) with SARS-CoV-2 RNA transcripts in singleplex reactions gBlocks DNA templates containing a 5’ T7 RNA polymerase promoter sequence were used to produce viral transcripts that corresponded to specific SARS-CoV-2 genomic regions (Fig. S1)
In the cases of SARS-CoV-2 L and S strains, specific TaqMan locked nucleic acid (LNA) probes were designed to discriminate between the single nucleotide polymorphism (SNP) found at position 8,782 of SARS-CoV-2 genomic sequence as described previously [23]
Summary
The human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) started in the Chinese city of Wuhan in late 2019, and spread around the world [1,2,3]. This novel coronavirus belongs to the subgroup betacoronavirus and the subgenus Sarbecovirus [3,4,5]. Thereafter, infected foreign residents of China and foreign travelers arriving in their countries of residence from international destinations, including visitors from Wuhan, contributed to the spread of the virus through person-to-person contact [4, 6, 7]. Spreading of the virus occurred and rapidly from person-to-person transmission in local communities, resulting in a pandemic spread across the globe
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