Department of Medical Biotechnology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran.
Department of Medical Biotechnology, Faculty of Medicine, Arak University of Medical Science, Arak, Iran.
Ir J Med Sci. 2023 Apr;192(2):723-729. doi: 10.1007/s11845-022-03046-2. Epub 2022 Jun 6.
In January 2020, the COVID-19 pandemic started and has severely affected all countries around the world. The clinical symptoms alone are not sufficient for a proper diagnosis. Thus, molecular tests are required. Various institutes and researchers developed real-time PCR-based methods for the detection of the virus. However, the method needs expensive equipment. In the present study, we developed a real-time NASBA assay for the detection of SARS-CoV-2.
Primers and molecular beacon probes for RdRp and N genes were designed. In silico analysis showed that primers and the probes were specific for SARS-CoV-2. The standard samples with known copy numbers of the virus were tested using the NASBA assay and an FDA-approved real-time PCR kit. A series of standard samples were prepared and tested. Clinical sensitivity, precision analysis, and clinical assessment of the assay were performed.
The limit of detection of the assay was 200 copies/mL. The clinical sensitivity of the assay was 97.64%. The intra-assay and inter-assay for both N and RdRp genes were less than 5% and 10%, respectively. Clinical assessment of the assay showed that the positive agreement rate and negative agreement rate of the assays were determined to be 97.64% and 100%, respectively.
The results of the present study show that the developed real-time NASBA is a sensitive and specific method for the detection of SARS-CoV-2 and is comparable with real-time PCR. NASBA is an isothermal signal amplification method, and if stand-alone fluorescent readers are available, the real-time NASBA can be used without the need for expensive thermocyclers. In addition compared to other isothermal methods like LAMP, the primer design is straightforward. Thus, real-time NASBA could be a suitable method for inexpensive SARS-CoV-2 detection.
2020 年 1 月，COVID-19 大流行开始，并严重影响了世界各国。仅凭临床症状不足以做出正确诊断。因此，需要进行分子检测。许多研究所和研究人员开发了基于实时 PCR 的方法来检测病毒。然而，该方法需要昂贵的设备。在本研究中，我们开发了一种用于检测 SARS-CoV-2 的实时 NASBA 检测法。
设计了 RdRp 和 N 基因的引物和分子信标探针。计算机分析表明，引物和探针对 SARS-CoV-2 具有特异性。使用 NASBA 检测法和经 FDA 批准的实时 PCR 试剂盒对具有已知病毒拷贝数的标准样本进行了测试。制备并测试了一系列标准样本。进行了该检测法的临床灵敏度、精密度分析和临床评估。
该检测法的检测限为 200 拷贝/ml。该检测法的临床灵敏度为 97.64%。N 和 RdRp 基因的内和间试验的精密度均小于 5%和 10%。该检测法的临床评估显示，该检测法的阳性符合率和阴性符合率分别为 97.64%和 100%。
本研究结果表明，开发的实时 NASBA 是一种用于检测 SARS-CoV-2 的敏感且特异的方法，与实时 PCR 相当。NASBA 是一种等温信号扩增方法，如果有独立的荧光读取器，无需昂贵的热循环仪即可使用实时 NASBA。此外，与其他等温方法（如 LAMP）相比，引物设计简单。因此，实时 NASBA 可能是一种用于低成本 SARS-CoV-2 检测的合适方法。
