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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

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 applicatio...

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Autores principales: Pham, Van Hung, Pham, Huong Thien, Balzanelli, Mario G., Distratis, Pietro, Lazzaro, Rita, Nguyen, Quoc Viet, Tran, Viet Quoc, Tran, Duy Khanh, Phan, Luan Duy, Pham, Sang Minh, Pham, Binh Thai, Duc, Chien Vo, Nguyen, Ha Minh, Nguyen, Dung Ngoc Thi, Tran, Ngoc Van, Pham, Son Truong, Queck, Camelia, Nguyen, Kieu Diem Cao, Inchingolo, Francesco, Del Prete, Raffaele, Nguyen, Nam Hai Dinh, Santacroce, Luigi, Gargiulo Isacco, Ciro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10137618/
https://www.ncbi.nlm.nih.gov/pubmed/37189465
http://dx.doi.org/10.3390/diagnostics13081364
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author Pham, Van Hung
Pham, Huong Thien
Balzanelli, Mario G.
Distratis, Pietro
Lazzaro, Rita
Nguyen, Quoc Viet
Tran, Viet Quoc
Tran, Duy Khanh
Phan, Luan Duy
Pham, Sang Minh
Pham, Binh Thai
Duc, Chien Vo
Nguyen, Ha Minh
Nguyen, Dung Ngoc Thi
Tran, Ngoc Van
Pham, Son Truong
Queck, Camelia
Nguyen, Kieu Diem Cao
Inchingolo, Francesco
Del Prete, Raffaele
Nguyen, Nam Hai Dinh
Santacroce, Luigi
Gargiulo Isacco, Ciro
author_facet Pham, Van Hung
Pham, Huong Thien
Balzanelli, Mario G.
Distratis, Pietro
Lazzaro, Rita
Nguyen, Quoc Viet
Tran, Viet Quoc
Tran, Duy Khanh
Phan, Luan Duy
Pham, Sang Minh
Pham, Binh Thai
Duc, Chien Vo
Nguyen, Ha Minh
Nguyen, Dung Ngoc Thi
Tran, Ngoc Van
Pham, Son Truong
Queck, Camelia
Nguyen, Kieu Diem Cao
Inchingolo, Francesco
Del Prete, Raffaele
Nguyen, Nam Hai Dinh
Santacroce, Luigi
Gargiulo Isacco, Ciro
author_sort Pham, Van Hung
collection PubMed
description 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.
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spelling pubmed-101376182023-04-28 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 Pham, Van Hung Pham, Huong Thien Balzanelli, Mario G. Distratis, Pietro Lazzaro, Rita Nguyen, Quoc Viet Tran, Viet Quoc Tran, Duy Khanh Phan, Luan Duy Pham, Sang Minh Pham, Binh Thai Duc, Chien Vo Nguyen, Ha Minh Nguyen, Dung Ngoc Thi Tran, Ngoc Van Pham, Son Truong Queck, Camelia Nguyen, Kieu Diem Cao Inchingolo, Francesco Del Prete, Raffaele Nguyen, Nam Hai Dinh Santacroce, Luigi Gargiulo Isacco, Ciro Diagnostics (Basel) Article 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. MDPI 2023-04-07 /pmc/articles/PMC10137618/ /pubmed/37189465 http://dx.doi.org/10.3390/diagnostics13081364 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Pham, Van Hung
Pham, Huong Thien
Balzanelli, Mario G.
Distratis, Pietro
Lazzaro, Rita
Nguyen, Quoc Viet
Tran, Viet Quoc
Tran, Duy Khanh
Phan, Luan Duy
Pham, Sang Minh
Pham, Binh Thai
Duc, Chien Vo
Nguyen, Ha Minh
Nguyen, Dung Ngoc Thi
Tran, Ngoc Van
Pham, Son Truong
Queck, Camelia
Nguyen, Kieu Diem Cao
Inchingolo, Francesco
Del Prete, Raffaele
Nguyen, Nam Hai Dinh
Santacroce, Luigi
Gargiulo Isacco, Ciro
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
title 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10137618/
https://www.ncbi.nlm.nih.gov/pubmed/37189465
http://dx.doi.org/10.3390/diagnostics13081364
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