MULTIPLEX SYBR GREEN ASSAY FOR CORONAVIRUS DETECTION USING FAST REAL-TIME RT-PCR

Salah & Et Al

doi:10.36103/ijas.v51i2.982

Abstract

This study was aimed to provide a local database for detection of coronavirus (CoV) species in suspect individual with respiratory tract infections like influenza type A and a tuberculosis using multiplex Sybr green reverse transcriptase real-time PCR (rRT-PCR) technique. A total of 500 samples was collected from individuals suffering from upper and/or lower respiratory tract diseases for testing of 4 CoV species (229E, OC43, NL63, and HKU1). RNA extracted, amplified and subsequent the positive samples sequencing. The results showed melting curve analysis (Tm) of the specific amplicons (79.73±0.36) and 9% positive for CoVs and some of them have other co-infection such as influenza virus 26.67%, and TB 11.11%. On the other hands, the CoVs were detected 4.62% in upper respiratory samples and 20.39% with lower respiratory samples. Sequencing results pointed out two isolates were CoV-NL63 and four isolates were CoV-229E, with first record accession number MN086823.1 and MN086824.1, respectively in GenBank. In conclusion, this rRT-PCR showed the rapid and efficient detection of CoVs with few copies number. This allows being used for the diagnosis of CoVs along with other respiratory viruses in a multiplex assay to reduce processing time. Subsequent applied nested RT-PCR to overcome the low viral load.

Highlights

Coronaviruses (CoVs) belonging to the family Coronaviridae in the order Nidovirales that infect the respiratory tract, a group of large enveloped RNA viruses between 70 and 120 nm in size, with a helical nucleocapsid and carries extended spike proteins on the membrane surface, providing the typical crown-like structure seen by electron microscopy, positive single-stranded with the largest genome of all known RNA viruses (27 to 33 kb) [30]
Both SARS-CoV and MERS-CoV is phylogenetically closely related to coronaviruses in bats [17]
This study aimed to provide a local database for detection of CoV species in suspect individual with respiratory tract infections like influenza type A and a tuberculosis using multiplex Sybr green Reverse transcriptionPolymerase Chain Reaction (rRT-PCR) technique

Summary

Introduction

Sybr green reverse transcriptase real-time PCR (rRTPCR) analysis for detecting HCoVs from acute respiratory tract infections because it has many benefits. This study aimed to provide a local database for detection of CoV species in suspect individual with respiratory tract infections like influenza type A and a tuberculosis using multiplex Sybr green rRT-PCR technique.

Objectives

Results

Conclusion