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. 2020 Jun;12(3):185-193.

Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR

Hamid Reza Mollaei  1 , Abass Aghaei Afshar  1 , Davood Kalantar-Neyestanaki  2 , Mehdi Fazlalipour  3 , Behnaz Aflatoonian  1
Affiliations

Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR

Hamid Reza Mollaei et al. Iran J Microbiol. 2020 Jun.

Abstract

Background and objectives: The new beta-coronavirus, which caused Severe Acute Respiratory Coronavirus-2 Syndrome (SARS-CoV-2), a major respiratory outbreak in Wuhan, China in December 2019, is now prevalent in many countries around the world. Identifying PCR-based viruses is a well-known and relatively stable protocol. Unfortunately, the high mutation rates may lead to widespread changes in viral nucleic acid sequences, and so using specific primers for PCR can be recommended. In this study, we evaluated the power of a conventional RT-PCR to detect SARS-CoV-2 RNA among the five set primer sets.

Materials and methods: The five genomic regions of the Coronavirus SARS-2 virus including Nucleocapsids (N), Envelope (E), RNA depended RNA Polymerase (RdRp), ORF1ab and Spike (S) were selected for primer designing. A conventional RT-PCR was performed to compare sensitivity, specificity and other analytical characteristics of primers designed against two Real Time PCR commercial kits.

Results: The result of the comparative analysis showed that the ORF1ab, N and RdRp primers had a sensitivity, specificity and positive predictive value higher than other primers. A significant difference in the analytical sensitivity between the studied primer sets in RT-PCR kits was observed.

Conclusion: In this study, the ORF1ab, Nucleocapsid and RdRp regions have the best primers for identifying the SARS-CoV-2 RNA between different genes that have been suggested.

Keywords: COVID-19; Coronavirus; Reverse transcription-polymerase chain reaction; Sars-CoV-2; Specific primer.

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Figures

Fig. 1.
Fig. 1.
Genome structure of SARS-2 (nCOVID-19) Gene N: Nucleocapsid phosphoprotein: 28274...29533, Gene E: Envelope: 26245...26472 Gene S: Spike: 21563...25384, ORF1ab:266…13468, 13468...21555, Gene M: Membrane glycoprotein: 26523...27191, RdRP:4393…5394
Fig. 2.
Fig. 2.
Gel electrophoresis of five products in 2% gel agarose, M: Marker 100 bp E gene; 145 bp, ORF1ab: 588 bp, RdRp: 196 bp, N gene: 323 bp, S gene: 440 bp
See this image and copyright information in PMC

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