A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions

被引：9

作者：

Chaintoutis, Serafeim C. ^{[1
]}

Chassalevris, Taxiarchis ^{[1
]}

Tsiolas, George ^{[2
]}

Balaska, Sofia ^{[3
]}

Vlatakis, Ioannis ^{[4
]}

Mouchtaropoulou, Evangelia ^{[2
]}

Siarkou, Victoria, I ^{[5
]}

Tychala, Areti ^{[3
]}

Koutsioulis, Dimitris ^{[4
]}

Skoura, Lemonia ^{[3
]}

Argiriou, Anagnostis ^{[2
,6
]}

Dovas, Chrysostomos, I ^{[1
]}

机构：

[1] Aristotle Univ Thessaloniki, Fac Hlth Sci, Sch Vet Med, Diagnost Lab, 11 Stavrou Voutyra Str, Thessaloniki 54627, Greece

[2] Ctr Res & Technol Hellas, Inst Appl Biosci, Thessaloniki 57001, Greece

[3] Aristotle Univ Thessaloniki, Ahepa Univ Hosp, Fac Hlth Sci, Med Sch,Dept Microbiol, 1 Stilponos Kyriakidi Str, Thessaloniki 54636, Greece

[4] EnzyQuest PC, Sci & Technol Pk Crete,100 Nikolaou Plastira Str, Iraklion 70013, Greece

[5] Aristotle Univ Thessaloniki, Fac Hlth Sci, Sch Vet Med, Lab Microbiol & Infect Dis, Univ Campus, Thessaloniki 54134, Greece

[6] Univ Aegean, Dept Food Sci & Nutr, Myrina 81400, Lemnos, Greece

来源：

JOURNAL OF VIROLOGICAL METHODS | 2021年 / 296卷

关键词：

SARS-CoV-2; Real-time RT-PCR; Mutation of concern; Typing; Spike protein;

D O I：

10.1016/j.jviromet.2021.114242

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94 % and a linear range of over 5 log(10) copies/reaction. Validation of the assay using known SARS-CoV-2-positive and negative samples from human and animals revealed its ability to correctly identify wild type strains, and strains possessing either one or both targeted amino-acid substitutions, thus comprising a useful pre-screening tool for rapid MOC identification. The basic principles of the methodology for the development of the assay are explained in order to facilitate the rapid design of similar assays able to detect emerging MOCs.

引用

页数：6

共 3 条

[1] Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with B.1.1.7 Variant of Concern
Abdulnoor, Mariana
Eshaghi, AliReza
Perusini, Stephen J.
Broukhanski, George
Corbeil, Antoine
Cronin, Kirby
Fittipaldi, Nahuel
Forbes, Jessica D.
Guthrie, Jennifer L.
Kus, Julianne, V
Li, Ye
Majury, Anna
Mallo, Gustavo, V
Mazzulli, Tony
Melano, Roberto G.
Olsha, Romy
Sullivan, Ashleigh
Tran, Vanessa
Patel, Samir N.
Allen, Vanessa G.
Gubbay, Jonathan B.
MICROBIOLOGY SPECTRUM, 2022, 10 (01):
[2] A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2
Dharavath, Bhasker
Yadav, Neelima
Desai, Sanket
Sunder, Roma
Mishra, Rohit
Ketkar, Madhura
Bhanshe, Prasanna
Gupta, Anurodh
Redhu, Archana Kumari
Patkar, Nikhil
Dutt, Shilpee
Gupta, Sudeep
Dutt, Amit
HELIYON, 2020, 6 (07)
[3] A Novel Real-Time RT-PCR-Based Methodology for the Preliminary Typing of SARS-CoV-2 Variants, Employing Non-Extendable LNA Oligonucleotides and Three Signature Mutations at the Spike Protein Receptor-Binding Domain
Chaintoutis, Serafeim C.
Chassalevris, Taxiarchis
Balaska, Sofia
Mouchtaropoulou, Evangelia
Tsiolas, George
Vlatakis, Ioannis
Tychala, Areti
Koutsioulis, Dimitris
Argiriou, Anagnostis
Skoura, Lemonia
Dovas, Chrysostomos, I
LIFE-BASEL, 2021, 11 (10):

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