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

Author(s):Show less -

Lesbon, Jessika Cristina Chagas ^[1] ; Poleti, Mirele Daiana ^[1] ; de Mattos Oliveira, Elisangela Chicaroni ^[1] ; Patane, Jose Salvatore Leister ^[2] ; Clemente, Luan Gaspar ^[3] ; Viala, Vincent Louis ^[2] ; Ribeiro, Gabriela ^[2] ; Giovanetti, Marta ^{[4, 5]} ; de Alcantara, Luiz Carlos Junior ^[5] ; de Lima, Loyze Paola Oliveira ^[2] ; Martins, Antonio Jorge ^[2] ; dos Santos Barros, Claudia Renata ^[2] ; Marqueze, Elaine Cristina ^[2] ; de Souza Todao Bernardino, Jardelina ^[2] ; Moretti, Debora Botequio ^[2] ; Brassaloti, Ricardo Augusto ^[3] ; de Lello Rocha Campos Cassano, Raquel ^[3] ; Mariani, Pilar Drummond Sampaio Correa ^[6] ; Slavov, Svetoslav Nanev ^[7] ; dos Santos, Rafael Bezerra ^[7] ; Rodrigues, Evandra Strazza ^[7] ; Santos, Elaine Vieira ^[7] ; Borges, Josiane Serrano ^[7] ; de La Roque, Debora Glenda Lima ^[7] ; Kitajima, Joao Paulo ^[8] ; Santos, Bibiana ^[8] ; Assato, Patricia Akemi ^[9] ; da Silva da Costa, Felipe Allan ^[9] ; Banho, Cecilia Artico ^[10] ; Sacchetto, Livia ^[10] ; Moraes, Marilia Mazzi ^[10] ; Palmieri, Melissa ^[11] ; da Silva, Fabiana Erica Vilanova ^[12] ; Grotto, Rejane Maria Tommasini ^[9] ; Souza-Neto, Jayme A. ^[9] ; Nogueira, Mauricio Lacerda ^[10] ; Coutinho, Luiz Lehman ^[3] ; Calado, Rodrigo Tocantins ^[7] ; Neto, Raul Machado ^[2] ; Covas, Dimas Tadeu ^[2] ; Kashima, Simone ^[7] ; Elias, Maria Carolina ^[2] ; Sampaio, Sandra Coccuzzo ^[2] ; Fukumasu, Heidge ^[1]

Affiliation:Show less -

^[1] Univ Sao Paulo, Sch Anim Sci & Food Engn, Dept Vet Med, Lab Comparat & Translat Oncol, BR-13635900 Pirassununga, SP - Brazil

^[2] Butantan Inst, BR-05503000 Sao Paulo, SP - Brazil

^[3] Univ Sao Paulo, Luiz de Queiroz Sch Agr, Dept Anim Sci, Funct Genom Ctr, BR-13418900 Piracicaba, SP - Brazil

^[4] Univ Fed Minas Gerais, Inst Biol Sci, BR-31270901 Belo Horizonte, MG - Brazil

^[5] Fiocruz MS, Fundacao Oswaldo Cruz, BR-21040900 Manguinho, RJ - Brazil

^[6] NGS Solucoes Genom, BR-13416 Piracicaba, SP - Brazil

^[7] Univ Sao Paulo, Blood Ctr Ribeirao Preto, Ribeirao Preto Med Sch, BR-14051 Ribeirao Preto, SP - Brazil

^[8] Mendel Genom Anal, BR-02511000 Sao Paulo, SP - Brazil

^[9] Sao Paulo State Univ, Sch Agr Sci, BR-18618970 Botucatu, SP - Brazil

^[10] Fac Med Sao Jose do Rio Preto, Dept Doencas Dermatol Infecciosas & Parasitarias, Lab Pesquisas Virol, BR-15090000 Sao Jose Do Rio Preto, SP - Brazil

^[11] Secretaria Municipal Saude, Coordenacao Vigilancia Saude, BR-01223906 Sao Paulo, SP - Brazil

^[12] Municipal Hlth Dept, Lab Assistance, Coordinat Primary Care, BR-01223906 Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: Viruses-Basel; v. 13, n. 12 DEC 2021.
• Web of Science Citations: 0

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. (AU)

• FAPESP's process: 20/10127-1 - Development of vaccine against COVID-19
• Grantee: Dimas Tadeu Covas
• Support Opportunities: Research Grants - Research in Public Policies
• FAPESP's process: 13/08135-2 - CTC - Center for Cell-Based Therapy
• Grantee: Dimas Tadeu Covas
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 20/05367-3 - Convalescent donor plasma to treat patients with severe SARS-CoV-2 infection (COVID-19)
• Grantee: Rodrigo do Tocantins Calado de Saloma Rodrigues
• Support Opportunities: Regular Research Grants