Rapid Homemade Reverse Transcriptase PCR Detection and Phylogenetic Analysis for SARS-CoV-2 Based on E and M genes

Abbas Morovvati; Ali Javadi; Somayeh Dehghani Sanij; Mohammad Reza Zarei; Shima Molazadeh; Mina  Badakhsh; Mohsen Zargar

doi:10.18502/jabs.v12i2.9883

Abbas Morovvati Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran
Ali Javadi Department of Pathobiology, Faculty of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Somayeh Dehghani Sanij Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran
Mohammad Reza Zarei Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran
Shima Molazadeh Department of Quality Assurance, Razi Vaccine & Serum Research Institute, Agricultural Research, Karaj, Iran
Mina Badakhsh Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran
Mohsen Zargar Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran

DOI: https://doi.org/10.18502/jabs.v12i2.9883

Keywords: RT-PCR, Covid-19, Detection, LOD, Phylogenetic Analysis

Abstract

Background & Objective: Coronavirus disease 2019 spreads worldwide and needs detection systems capable of rapid diagnostic of this virus (SARS-CoV-2). The aim of this study is to design the homemade RT-PCR method for the Detection and phylogenetic analysis of this virus.

Material & Methods: The genes selected for diagnosis were E and M genes for this virus. PCR product was cloned in pTZ57R/T plasmid for preparation of positive control. In order to determine the sensitivity of this molecular method, the genes mentioned in the clone pTZ57R/T vector and the Limit of detection (LOD) the genes were determined and phylogenetic analysis was performed using partial E and M gene sequences.

Results: PCR product was observed for E and M genes 156 and 547 bp on the Agarose gel. The LOD of the E and M gene was 60 and 82 copies. There was also a positive response to the samples of patients who were positive by other methods.

Conclusions: Since this virus is considered to be the cause of a pandemic in different countries all over the world, the present study is very important as a method of rapid and low-cost molecular diagnosis for monitoring this virus. Phylogenetic analysis is necessary for epidemiological studies for the control and prevention of the disease.