A low-cost TaqMan minor groove binder probe-based one-step RT-qPCR assay for rapid identification of N501Y variants of SARS-CoV-2.

Autor: Chan CT; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region., Leung JS; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region., Lee LK; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region., Lo HW; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region., Wong EY; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region., Wong DS; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region., Ng TT; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region., Lao HY; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region., Lu KK; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region., Jim SH; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region., Yau MC; Department of Clinical Pathology, Pamela Youde Nethersole Eastern Hospital, Hong Kong Special Administrative Region., Lam JY; Department of Clinical Pathology, Pamela Youde Nethersole Eastern Hospital, Hong Kong Special Administrative Region., Ho AY; Department of Pathology, Princess Margaret Hospital, Hong Kong Special Administrative Region., Luk KS; Department of Pathology, Princess Margaret Hospital, Hong Kong Special Administrative Region., Yip KT; Department of Clinical Pathology, Tuen Mun Hospital, Hong Kong Special Administrative Region., Que TL; Department of Clinical Pathology, Tuen Mun Hospital, Hong Kong Special Administrative Region., To KK; Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region., Siu GK; Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region. Electronic address: gilman.siu@polyu.edu.hk.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2022 Jan; Vol. 299, pp. 114333. Date of Electronic Publication: 2021 Oct 14.
DOI: 10.1016/j.jviromet.2021.114333
Abstrakt: The increasing prevalence of N501Y variants of SARS-CoV-2 has kindled global concern due to their enhanced transmissibility. Genome sequencing is the gold standard method to identify the emerging variants of concern. But it is time-consuming and expensive, limiting the widespread deployment of genome surveillance in some countries. Health authorities surge the development of alternative assay to expand screening capacity with reduced time and cost. In this study, we developed an in-house TaqMan minor groove binder (MGB) probe-based one-step RT-qPCR assay to detect the presence of N501Y mutation in SARS-CoV-2. A total of 168 SARS-CoV-2 positive respiratory specimens were collected to determine diagnostic accuracy of the RT-qPCR assay. As a reference standard, PANGO lineages and the mutation patterns of all samples were characterised by whole-genome sequencing. The analytical sensitivity and the ability of the assay to detect low frequency of N501Y variants were also evaluated. A total of 31 PANGO lineages were identified from 168 SARS-CoV-2 positive cases, in which 34 samples belonged to N501Y variants, including B.1.1.7 (n = 20), B.1.351 (n = 12) and P.3 (n = 2). The N501Y RT-qPCR correctly identified all 34 samples as N501Y-positive and the other 134 samples as wildtype. The limit-of-detection of the assay consistently achieved 1.5 copies/μL on four different qPCR platforms. N501Y mutation was successfully detected at an allele frequency as low as 10 % in a sample with mixed SARS-CoV-2 lineage. The N501Y RT-qPCR is simple and inexpensive (US$1.6 per sample). It enables robust high-throughput screening for surveillance of SARS-CoV-2 variants of concern harbouring N501Y mutation.
(Copyright © 2021 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: