描述了严重急性呼吸综合征相关冠状病毒2(SARS-CoV-2)的多重分子诊断测定,甲型(IAV)和乙型(IBV)流感病毒主要基于实时反应,这限制了他们进入许多实验室或诊断机构。有助于现有的策略和扩大获得鉴别诊断,我们描述了针对SARS-CoV-2,IAV和IBV的同时内源性对照扩增的终点多重RT-PCR。最初,我们寻找SARS-CoV-2,IAV,IBV和RNA酶P,其扩增子可以在琼脂糖凝胶上区分。然后通过优化反应混合物和循环条件将多重测定标准化。检测限(LoD)使用滴定的病毒(对于SARS-CoV-2和IAV)并通过从IBV阳性样品池中稀释来确定。通过测试具有不同RNA酶P和病毒载量的样品来评估多重诊断性能,先前鉴定为目标病毒阳性或阴性。IAV的扩增子(146bp),SARS-CoV-2(113bp),IBV(103bp)和RNA酶P(65bp)在我们的多重分析中得到了充分的区分。SARS-CoV-2、IAV和IBV的LoD为0.02TCID50/ml,0.07TCID50/ml和10-3来自阳性样品池,分别。所有样本均为SARS-CoV-2阳性(n=70,Ct17.2-36.9),在我们的多重测定中,IAV(n=53,Ct14-34.9)和IBV(n=12,Ct23.9-31.9)保持阳性。来自阴性样品(n=40,Ct25.2-30.2)的RNA酶P也在多重中扩增。总的来说,我们的检测是一种及时的替代工具,用于在供应/设备有限的实验室中检测SARS-CoV-2和流感病毒.
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.