Evaluation of RT-PCR assays for detection of SARS-CoV-2 variants of concern.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic has been considered with great importance on correct screening procedure. The detection efficiency of recent variants of concern were observed by comparing 5 commercial RT-PCR kits and a SYBR-green method developed and validated in our laboratory. The RNA was extracted from nasopharyngeal samples from suspected COVID-19 patients and RT-PCR assay was performed according to the instruction of the respective manufacturers. The specificity and sensitivity of Maccura kit was 81.8% and 82.5%, A*Star kit was 100% and 75.4%, Da An Gene kit was 100% and 68.4%, Sansure kit was 54.5% and 91.2% and TaqPath kit was 100% and 70.2% respectively. Our in house SYBR-Green method showed a consistent detection result with 90.9% specificity and 91.2% sensitivity. We also found that detection kits targeting more genes showed better accuracy which facilitates less false positive results (< 20%). Our study found a significant difference (p < 0.005) in Ct value reported for common target genes shared by the RT-PCR kits in relation with different variants of COVID-19 infection. Recent variants of concerns contain more than 30 mutations in the spike proteins including 2 deletion and a unique insertion mutation by which makes detection of these variants difficult and these facilitates the variants to escape from being detected.

Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis.

TaqMan probe-based commercial real-time (RT) PCR kits are expensive but most frequently used in COVID-19 diagnosis. The unprecedented scale of SARS-CoV-2 infections needs to meet the challenge of testing more persons at a reasonable cost. This study developed a simple and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay targeting two virus-specific genes along with a host-specific internal control. A total of 180 randomly selected samples portioning into two subsets based on crude and high-quality RNA extraction were used to compare this assay with a nationwide available commercial kit (Sansure Biotech Inc., (Hunan, China)), so that we could analyze the variation and validity of this in-house developed method. Our customized-designed primers can specifically detect the viral RNA likewise Sansure. We separately optimized SYBR Green RT-PCR reaction of N, E, S, and RdRp genes based on singleplex melting curve analysis at the initial stage. After several rounds of optimization on multiplex assays of different primer combinations, the optimized method finally targeted N and E genes of the SARS-CoV-2 virus, together with the ß-actin gene of the host as an internal control. Comparing with the Sansure commercial kit, our proposed assay provided up to 97% specificity and 93% sensitivity. The cost of each sample processing ranged between ~2 and ~6 USD depending on the purification level of extracted RNA template. Overall, this one-step and one-tube method can revolutionize the COVID-19 diagnosis in low-income countries.