Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results.

Autor:	Lesbon JCC; 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., Poleti MD; 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., de Mattos Oliveira EC; 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., Patané JSL; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Clemente LG; Functional Genomic Center, Department of Animal Science, Luiz de Queiroz School of Agriculture, University of Sao Paulo, Piracicaba 13418-900, São Paulo, Brazil., Viala VL; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Ribeiro G; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Giovanetti M; Fundação Oswaldo Cruz, FIOCRUZ, Manguinhos 21040-900, Rio de Janeiro, Brazil.; Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte 31270-901, Minas Gerais, Brazil., de Alcantara LCJ; Fundação Oswaldo Cruz, FIOCRUZ, Manguinhos 21040-900, Rio de Janeiro, Brazil., Teixeira O; Ribeirao Preto Protein Crystallography Laboratory, School of Pharmaceutical Sciences, University of São Paulo, Ribeirao Preto 14040-903, São Paulo, Brazil, Nonato MC; Ribeirao Preto Protein Crystallography Laboratory, School of Pharmaceutical Sciences, University of São Paulo, Ribeirao Preto 14040-903, São Paulo, Brazil, de Lima LPO; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Martins AJ; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Dos Santos Barros CR; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Marqueze EC; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., de Souza Todão Bernardino J; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Moretti DB; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Brassaloti RA; Functional Genomic Center, Department of Animal Science, Luiz de Queiroz School of Agriculture, University of Sao Paulo, Piracicaba 13418-900, São Paulo, Brazil., de Lello Rocha Campos Cassano R; Functional Genomic Center, Department of Animal Science, Luiz de Queiroz School of Agriculture, University of Sao Paulo, Piracicaba 13418-900, São Paulo, Brazil., Mariani PDSC; NGS Soluções Genômicas, Piracicaba 13416-030, São Paulo, Brazil., Slavov SN; Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil, Dos Santos RB; Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil, Rodrigues ES; Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil, Santos EV; Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil, Borges JS; Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil, de La Roque DGL; Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil, Kitajima JP; Mendelics Genomic Analysis, São Paulo 02511-000, São Paulo, Brazil., Santos B; Mendelics Genomic Analysis, São Paulo 02511-000, São Paulo, Brazil., Assato PA; School of Agricultural Sciences, São Paulo State University, Botucatu 18618-970, São Paulo, Brazil., da Silva da Costa FA; School of Agricultural Sciences, São Paulo State University, Botucatu 18618-970, São Paulo, Brazil., Banho CA; 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., Sacchetto L; 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., Moraes MM; 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., Palmieri M; Coordenação de Vigilância em Saúde-Secretaria Municipal da Saúde, São Paulo, São Paulo 01223-906, Brazil., da Silva FEV; Laboratory Assistance, Coordination of Primary Care, Municipal Health Department, São Paulo, São Paulo 01223-906, Brazil., Grotto RMT; School of Agricultural Sciences, São Paulo State University, Botucatu 18618-970, São Paulo, Brazil., Souza-Neto JA; School of Agricultural Sciences, São Paulo State University, Botucatu 18618-970, São Paulo, Brazil., Nogueira ML; 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., Coutinho LL; Functional Genomic Center, Department of Animal Science, Luiz de Queiroz School of Agriculture, University of Sao Paulo, Piracicaba 13418-900, São Paulo, Brazil., Calado RT; Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil, Neto RM; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Covas DT; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil.; Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil, Kashima S; Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, São Paulo, Brazil, Elias MC; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Sampaio SC; Butantan Institute, São Paulo 05503-000, São Paulo, Brazil., Fukumasu H; 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.
Jazyk:	angličtina
Zdroj:	Viruses [Viruses] 2021 Dec 10; Vol. 13 (12). Date of Electronic Publication: 2021 Dec 10.
DOI:	10.3390/v13122474
Abstrakt:	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.
Databáze:	MEDLINE
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