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© 2021 by the authors. 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/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.

Details

Title
Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results
Author
Jéssika Cristina Chagas Lesbon 1   VIAFID ORCID Logo  ; Poleti, Mirele Daiana 1 ; Elisângela Chicaroni de Mattos Oliveira 1   VIAFID ORCID Logo  ; José Salvatore Leister Patané 2 ; Luan Gaspar Clemente 3   VIAFID ORCID Logo  ; Viala, Vincent Louis 2   VIAFID ORCID Logo  ; Ribeiro, Gabriela 2 ; Giovanetti, Marta 4 ; Luiz Carlos Junior de Alcantara 5 ; Teixeira, Olivia 6   VIAFID ORCID Logo  ; Nonato, Maria Cristina 6   VIAFID ORCID Logo  ; Loyze Paola Oliveira de Lima 2 ; Martins, Antonio Jorge 2 ; Claudia Renata dos Santos Barros 2   VIAFID ORCID Logo  ; Marqueze, Elaine Cristina 2   VIAFID ORCID Logo  ; Jardelina de Souza Todão Bernardino 2 ; Moretti, Debora Botequio 2   VIAFID ORCID Logo  ; Brassaloti, Ricardo Augusto 3   VIAFID ORCID Logo  ; Raquel de Lello Rocha Campos Cassano 3 ; Pilar Drummond Sampaio Correa Mariani 7 ; Slavov, Svetoslav Nanev 8   VIAFID ORCID Logo  ; Rafael Bezerra dos Santos 8 ; Evandra Strazza Rodrigues 8   VIAFID ORCID Logo  ; Elaine Vieira Santos 8 ; Josiane Serrano Borges 8 ; Debora Glenda Lima de La Roque 8   VIAFID ORCID Logo  ; Kitajima, Joao Paulo 9 ; Santos, Bibiana 9 ; Patricia Akemi Assato 10 ; Felipe Allan da Silva da Costa 10 ; Cecilia Artico Banho 11   VIAFID ORCID Logo  ; Sacchetto, Livia 11   VIAFID ORCID Logo  ; Marilia Mazzi Moraes 11   VIAFID ORCID Logo  ; Palmieri, Melissa 12 ; Fabiana Erica Vilanova da Silva 13 ; Rejane Maria Tommasini Grotto 10   VIAFID ORCID Logo  ; Souza-Neto, Jayme A 10   VIAFID ORCID Logo  ; Mauricio Lacerda Nogueira 11 ; Luiz Lehman Coutinho 3 ; Calado, Rodrigo Tocantins 8   VIAFID ORCID Logo  ; Raul Machado Neto 2 ; Dimas Tadeu Covas 14 ; Kashima, Simone 8 ; Elias, Maria Carolina 2   VIAFID ORCID Logo  ; Sampaio, Sandra Coccuzzo 2 ; Fukumasu, Heidge 1   VIAFID ORCID Logo 

 Laboratory of Comparative and Translational Oncology, Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, São Paulo, Brazil; [email protected] (J.C.C.L.); [email protected] (M.D.P.); [email protected] (E.C.d.M.O.) 
 Butantan Institute, São Paulo 05503-000, São Paulo, Brazil; [email protected] (J.S.L.P.); [email protected] (V.L.V.); [email protected] (G.R.); [email protected] (L.P.O.d.L.); [email protected] (A.J.M.); [email protected] (C.R.d.S.B.); [email protected] (E.C.M.); [email protected] (J.d.S.T.B.); [email protected] (D.B.M.); [email protected] (R.M.N.); [email protected] (D.T.C.); [email protected] (M.C.E.); [email protected] (S.C.S.) 
 Functional Genomic Center, Department of Animal Science, Luiz de Queiroz School of Agriculture, University of Sao Paulo, Piracicaba 13418-900, São Paulo, Brazil; [email protected] (L.G.C.); [email protected] (R.A.B.); [email protected] (R.d.L.R.C.C.); [email protected] (L.L.C.) 
 Fundação Oswaldo Cruz, FIOCRUZ, Manguinhos 21040-900, Rio de Janeiro, Brazil; [email protected] (M.G.); [email protected] (L.C.J.d.A.); Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte 31270-901, Minas Gerais, Brazil 
 Fundação Oswaldo Cruz, FIOCRUZ, Manguinhos 21040-900, Rio de Janeiro, Brazil; [email protected] (M.G.); [email protected] (L.C.J.d.A.) 
 Ribeirao Preto Protein Crystallography Laboratory, School of Pharmaceutical Sciences, University of São Paulo, Ribeirao Preto 14040-903, São Paulo, Brazil; [email protected] (O.T.); [email protected] (M.C.N.) 
 NGS Soluções Genômicas, Piracicaba 13416-030, São Paulo, Brazil; [email protected] 
 Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil; [email protected] (S.N.S.); [email protected] (R.B.d.S.); [email protected] (E.S.R.); [email protected] (E.V.S.); [email protected] (J.S.B.); [email protected] (D.G.L.d.L.R.); [email protected] (R.T.C.); [email protected] (S.K.) 
 Mendelics Genomic Analysis, São Paulo 02511-000, São Paulo, Brazil; [email protected] (J.P.K.); [email protected] (B.S.) 
10  School of Agricultural Sciences, São Paulo State University, Botucatu 18618-970, São Paulo, Brazil; [email protected] (P.A.A.); [email protected] (F.A.d.S.d.C.); [email protected] (R.M.T.G.); [email protected] (J.A.S.-N.) 
11  Laboratório de Pesquisas em Virologia, Departamento de Doenças Dermatológicas, Infecciosas e Parasitárias, Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto 15090-000, São Paulo, Brazil; [email protected] (C.A.B.); [email protected] (L.S.); [email protected] (M.M.M.); [email protected] (M.L.N.) 
12  Coordenação de Vigilância em Saúde—Secretaria Municipal da Saúde, São Paulo 01223-906, São Paulo, Brazil; [email protected] 
13  Laboratory Assistance, Coordination of Primary Care, Municipal Health Department, São Paulo 01223-906, São Paulo, Brazil; [email protected] 
14  Butantan Institute, São Paulo 05503-000, São Paulo, Brazil; [email protected] (J.S.L.P.); [email protected] (V.L.V.); [email protected] (G.R.); [email protected] (L.P.O.d.L.); [email protected] (A.J.M.); [email protected] (C.R.d.S.B.); [email protected] (E.C.M.); [email protected] (J.d.S.T.B.); [email protected] (D.B.M.); [email protected] (R.M.N.); [email protected] (D.T.C.); [email protected] (M.C.E.); [email protected] (S.C.S.); Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil; [email protected] (S.N.S.); [email protected] (R.B.d.S.); [email protected] (E.S.R.); [email protected] (E.V.S.); [email protected] (J.S.B.); [email protected] (D.G.L.d.L.R.); [email protected] (R.T.C.); [email protected] (S.K.) 
First page
2474
Publication date
2021
Year
2021
Publisher
MDPI AG
e-ISSN
19994915
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2612844817
Copyright
© 2021 by the authors. 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/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.