Development of DMSc COVID-19 real-time RT-PCR
Keywords:
COVID-19, SARS-CoV-2, real-time RT-PCRAbstract
Development of diagnostic tests for detection of emerging infectious agents in patients, contact cases and high-risk group is critical for disease control and early effective treatment. In this study, we aimed to develop DMSc COVID-19 real-time RT-PCR assay for detection of RdRp and N gene which are specific for SARS-CoV-2 causing COVID-19, to evaluate a simplified extraction-free methods (heat and direct lysis) and to evaluate effects of five enhancers; Tween 20 (0.5%), Glycerol (10, 15, 20%), Dimethyl sulfoxide (DMSO) (10%), Bovine serum albumin (BSA) (10 mg/mL) and Triton X-100 (0.1%) on amplification efficiency. The results demonstrated that the real-time RT-PCR assay had analytical sensitivity to detect RdRp and N gene at 50 and 5 virus copies per reaction, respectively. Among extraction-free methods, heat and direct lysis showed diagnostic sensitivity of 97.14 and 100% and specificity of 95.89 and 94.52% respectively. Enhancer mixes composed of Tween 20 (0.5%) and Glycerol (15%) increased diagnostic sensitivity and specificity up to 100%. Our study revealed that the DMSc COVID-19 real-time RT-PCR assay could be used to amplify RNA without extraction process. This assay could serve as a rapid and cost-effective diagnostic method to increase capacity of COVID-19 detection for monitoring and diagnostic purposes.
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