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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.
- Received:
- Accepted:
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Funding
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
- Principle Award Recipient: ThaísaRegina Rocha Lopes
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Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul
(Award 20/2551-0000252-7)
- Principle Award Recipient: ThaísaRegina Rocha Lopes
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Conselho Nacional de Desenvolvimento Científico e Tecnológico
(Award 303981/2022-9)
- Principle Award Recipient: RudiWeiblen
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Conselho Nacional de Desenvolvimento Científico e Tecnológico
(Award 301414/2010-6)
- Principle Award Recipient: EduardoFurtado Flores
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Conselho Nacional de Desenvolvimento Científico e Tecnológico
- Principle Award Recipient: JoséValter Joaquim Silva Júnior
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Conselho Nacional de Desenvolvimento Científico e Tecnológico
- Principle Award Recipient: ThaísaRegina Rocha Lopes