An end-point multiplex RT-PCR for SARS-CoV-2, Influenza A and B detection, including simultaneous RNAse P amplification: a timely tool for more accessible differential diagnosis.

Lopes, Thaísa Regina Rocha; Silva Júnior, José Valter Joaquim; Trindade, Priscila de Arruda; Gregianini, Tatiana Schäffer; Weiblen, Rudi; Flores, Eduardo Furtado

Lopes, Thaísa Regina Rocha; Silva Júnior, José Valter Joaquim; Trindade, Priscila de Arruda; Gregianini, Tatiana Schäffer; Weiblen, Rudi; Flores, Eduardo Furtado.

Afiliação

Lopes TRR; Programa de Pós-graduação em Medicina Veterinária, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.
Silva Júnior JVJ; Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.
Trindade PA; Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.
Gregianini TS; Setor de Virologia, Instituto Keizo Asami, Universidade Federal de Pernambuco, Pernambuco, Brazil.
Weiblen R; Laboratório NB3 de Neuroimunologia, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.
Flores EF; Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.

J Med Microbiol ; 73(8)2024 Aug.

Article em En | MEDLINE | ID: mdl-39140993

ABSTRACT

ABSTRACT

The multiplex molecular diagnostic assays described for severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), influenza A (IAV) and B (IBV) viruses have been mainly based on real-time reaction, which limits their access to many laboratories or diagnostic institutions. To contribute to available strategies and expand access to differential diagnosis, we describe an end-point multiplex RT-PCR targeting SARS-CoV-2, IAV and IBV with simultaneous endogenous control amplification. Initially, we looked for well-established primers sets for SARS-CoV-2, IAV, IBV and RNAse P whose amplicons could be distinguished on agarose gel. The multiplex assay was then standardized by optimizing the reaction mix and cycle conditions. The limit of detection (LoD) was determined using titrated viruses (for SARS-CoV-2 and IAV) and by dilution from a pool of IBV-positive samples. The diagnostic performance of the multiplex was evaluated by testing samples with different RNAse P and viral loads, previously identified as positive or negative for the target viruses. The amplicons of IAV (146 bp), SARS-CoV-2 (113 bp), IBV (103 bp) and RNAse P (65 bp) were adequately distinguished in our multiplex. The LoD for SARS-CoV-2, IAV and IBV was 0.02 TCID50/ml, 0.07 TCID50/ml and 10-3 from a pool of positive samples, respectively. All samples positive for SARS-CoV-2 (n=70, Ct 17.2-36.9), IAV (n=53, Ct 14-34.9) and IBV (n=12, Ct 23.9-31.9) remained positive in our multiplex assay. RNAse P from negative samples (n=40, Ct 25.2-30.2) was also amplified in the multiplex. Overall, our assay is a timely and alternative tool for detecting SARS-CoV-2 and influenza viruses in laboratories with limited access to supplies/equipment.

Assuntos

COVID-19; Vírus da Influenza A; Vírus da Influenza B; Reação em Cadeia da Polimerase Multiplex; Ribonuclease P; SARS-CoV-2; Humanos; Ribonuclease P/genética; SARS-CoV-2/genética; SARS-CoV-2/isolamento & purificação; Vírus da Influenza A/isolamento & purificação; Vírus da Influenza A/genética; Vírus da Influenza B/isolamento & purificação; Vírus da Influenza B/genética; COVID-19/diagnóstico; COVID-19/virologia; Reação em Cadeia da Polimerase Multiplex/métodos; Diagnóstico Diferencial; Influenza Humana/diagnóstico; Influenza Humana/virologia; Sensibilidade e Especificidade; Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos; Limite de Detecção; RNA Viral/genética; RNA Viral/análise

Palavras-chave

coronavirus disease 2019; differential diagnosis; flu; molecular diagnosis

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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Vírus da Influenza A / Vírus da Influenza B / Ribonuclease P / Reação em Cadeia da Polimerase Multiplex / SARS-CoV-2 / COVID-19 Limite: Humans Idioma: En Revista: J Med Microbiol Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Brasil País de publicação: Reino Unido

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