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

2021; Multidisciplinary Digital Publishing Institute; Volume: 13; Issue: 12 Linguagem: Inglês

10.3390/v13122474

1999-4915

Jéssika Cristina Chagas Lesbon, Mirele Poleti, E. C. Mattos, José Salvatore Leister Patané, Luan Gaspar Clemente, Vincent Louis Viala, Gabriela Ribeiro, Marta Giovanetti, Luíz Carlos Júnior Alcântara, Olívia Teixeira, Maria Cristina Nonato, Luciana Lima, Antonio Jorge Martins, Cláudia Renata dos Santos Barros, Elaine Cristina Marqueze, Jardelina de Souza Todão Bernardino, Débora Botéquio Moretti, Ricardo Augusto Brassaloti, Raquel de Lello Rocha Campos Cassano, Pilar Mariani, Svetoslav Nanev Slavov, Rafael dos Santos, Evandra Strazza Rodrigues, Elaine Vieira Santos, Jéssica Bassani Borges, Debora Glenda Lima de La‐Roque, João Paulo Kitajima, Bibiana Santos, Patrícia Akemi Assato, Felipe Allan da Silva da Costa, Cecília Ártico Banho, Lívia Sacchetto, Marília Mazzi Moraes, Melissa Palmieri, Fabiana da Silva, Rejane Maria Tommasini Grotto, Jayme A. Souza‐Neto, Maurício Lacerda Nogueira, Luiz Lehmann Coutinho, Rodrigo T. Calado, Raul Machado Neto, Dimas Tadeu Covas, Simone Kashima, Maria Carolina Elias, Sandra Coccuzzo Sampaio, Heidge Fukumasu,

SARS-CoV-2 detection and testing

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.