Multiplex Sybr green assay for coronavirus detection using fast real-time RT-PCR

Abstract EN

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.