| Autor: |
Lopes TRR; Programa de Pós-graduação em Medicina Veterinária, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.; Setor de Virologia, Departamento de Medicina Veterinária Preventiva, 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.; Setor de Virologia, Instituto Keizo Asami, Universidade Federal de Pernambuco, Pernambuco, Brazil.; Laboratório NB3 de Neuroimunologia, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.; Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil., Trindade PA; Laboratório de Biologia Molecular e Bioinformática Aplicada à Microbiologia Clínica, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil., Gregianini TS; Laboratório Central de Saúde, Centro Estadual de Vigilância em Saúde da Secretaria de Saúde do estado do Rio Grande do Sul, Rio Grande do Sul, Brazil., Weiblen R; Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil., Flores EF; Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil. |
| Abstrakt: |
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 TCID 50 /ml, 0.07 TCID 50 /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. |
