Detection of SARS-CoV-2 using five primer sets

Abstract

A novel coronavirus (SARS-CoV-2) outbreak, responsible for a pneumonia-associated respiratory disorder (COVID-19), has started in early December 2019 in Wuhan, China, and has rapidly spread around the world. Rapid and accurate diagnostic testing plays a crucial role in tackling the COVID-19 pandemic. In this study, it was aimed to compare 5 primer sets designed to amplify different regions for the detection of SARS-CoV-2 and to perform sequence analysis. Conventional RT-PCR was carried out using primers targeting different regions of the virus genome including ORF1ab, Envelope (E), RNA-dependent RNA polymerase (RdRp), Spike (S) and Nucleocapsid (N) genes for the diagnosis of COVID-19. DNA sequence of ORF1ab gene from each sample were compared with the DNA sequence data of SARS-CoV-2 stored in the GenBank and ORF1ab phylogenetic tree was constructed. The amplicon sizes of ORF1ab, S, E, N and RdRp genes were 588 bp, 440 bp, 145 bp, 323 bp and 196 bp, respectively. The 2019-nCoV RNA was detected from 74% of total samples from RdRp gene, 87% for N gene, 74% for S gene, 61% for E gene and 82% for ORF1ab region. The ORF1ab sequences of SARS-CoV-2 from 82 patients were had 100% identity to the sequence of Wuhan isolate and among themselves. The phylogenetic analysis revealed that all isolates formed a cluster. The results of this study suggest that the N region is the best for SARS-CoV-2 identification.

Comparison of analytical sensitivity and efficiency for SARS-CoV-2 primer sets by TaqMan-based and SYBR Green-based RT-qPCR

“…Although mismatches have been revealed in N, 2019-nCoV_N1, 2019-nCoV_N3, NIID_2019-nCoV_2, and WH-NIC N previously (Alvarez-Diaz et al 2020 ; Elaswad et al 2020 ; Khan and Cheung 2020b ) and in this study, suggesting the N gene may be under positive selective pressure where many mutations accumulates (Elaswad et al 2020 ; Lo Presti et al 2020 ); the N region has been still widely considered as the best and most commonly used for SARS-CoV-2 identification (Karagöz et al 2020 ; Mollaei et al 2020 ).…”

“…ORF1 which included RdRp region was recommended as one of the best regions for SARS-CoV-2 identification besides the N region (Karagöz et al 2020 ; Mollaei et al 2020 ). Yet, positive selection has been demonstrated for specific residues of the non-structural proteins of ORF1ab and the accessory proteins ORF3a and ORF8 which might affect PCR test accuracy (Velazquez-Salinas et al 2020 ).…”