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Multiplex RT Real-Time PCR Based on Target Failure to Detect and Identify Different Variants of SARS-CoV-2: A Feasible Method That Can Be Applied in Clinical Laboratories

Authors :
Van Hung Pham
Huong Thien Pham
Mario G. Balzanelli
Pietro Distratis
Rita Lazzaro
Quoc Viet Nguyen
Viet Quoc Tran
Duy Khanh Tran
Luan Duy Phan
Sang Minh Pham
Binh Thai Pham
Chien Vo Duc
Ha Minh Nguyen
Dung Ngoc Thi Nguyen
Ngoc Van Tran
Son Truong Pham
Camelia Queck
Kieu Diem Cao Nguyen
Francesco Inchingolo
Raffaele Del Prete
Nam Hai Dinh Nguyen
Luigi Santacroce
Ciro Gargiulo Isacco
Source :
Diagnostics, Vol 13, Iss 8, p 1364 (2023)
Publication Year :
2023
Publisher :
MDPI AG, 2023.

Abstract

Shortly after its emergence, Omicron and its sub-variants have quickly replaced the Delta variant during the current COVID-19 outbreaks in Vietnam and around the world. To enable the rapid and timely detection of existing and future variants for epidemiological surveillance and diagnostic applications, a robust, economical real-time PCR method that can specifically and sensitively detect and identify multiple different circulating variants is needed. The principle of target- failure (TF) real-time PCR is simple. If a target contains a deletion mutation, then there is a mismatch with the primer or probe, and the real-time PCR will fail to amplify the target. In this study, we designed and evaluated a novel multiplex RT real-time PCR (MPL RT-rPCR) based on the principle of target failure to detect and identify different variants of SARS-CoV-2 directly from the nasopharyngeal swabs collected from COVID-19 suspected cases. The primers and probes were designed based on the specific deletion mutations of current circulating variants. To evaluate the results from the MPL RT-rPCR, this study also designed nine pairs of primers for amplifying and sequencing of nine fragments from the S gene containing mutations of known variants. We demonstrated that (i) our MPL RT-rPCR was able to accurately detect multiple variants that existed in a single sample; (ii) the limit of detection of the MPL RT-rPCR in the detection of the variants ranged from 1 to 10 copies for Omicron BA.2 and BA.5, and from 10 to 100 copies for Delta, Omicron BA.1, recombination of BA.1 and BA.2, and BA.4; (iii) between January and September 2022, Omicron BA.1 emerged and co-existed with the Delta variant during the early period, both of which were rapidly replaced by Omicron BA.2, and this was followed by Omicron BA.5 as the dominant variant toward the later period. Our results showed that SARS-CoV-2 variants rapidly evolved within a short period of time, proving the importance of a robust, economical, and easy-to-access method not just for epidemiological surveillance but also for diagnoses around the world where SARS-CoV-2 variants remain the WHO’s highest health concern. Our highly sensitive and specific MPL RT-rPCR is considered suitable for further implementation in many laboratories, especially in developing countries.

Details

Language :
English
ISSN :
20754418
Volume :
13
Issue :
8
Database :
Directory of Open Access Journals
Journal :
Diagnostics
Publication Type :
Academic Journal
Accession number :
edsdoj.7eafb5ea1e6e450d98fbd10ec1e904f6
Document Type :
article
Full Text :
https://doi.org/10.3390/diagnostics13081364