Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes

Huseyin Tombuloglu,Moneerah Alsaeed,Ebtesam Al-Suhaimi,Hamoud Al-Khallaf,Juma H Kabanja,Najat Al-Saleh,Hussein Sabit

doi:10.1038/s41598-022-06977-z

Abstract

Corona Virus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative sensitivity, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, due to the rapid mutation rate of the viral genome and the emergence of new variants, existing protocols need to be updated and improved. Designing a fast and accurate PCR-based assay is of great importance for the early detection of this virus and more efficient control of the spread of this disease. This study describes a fast, reliable, easy-to-use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N and RdRP) and a human gene (RP) simultaneously. The performance and the sensitivity of the assay were tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.40 and 0.81 copies/µL or 35.13 and 20.31 copies/reaction for RdRP and N genes, respectively. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Highlights

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the subfamily Orthocoronavirinae of the family Coronaviridae of the order Nidovirales
A multiplex rRT-polymerase chain reaction (PCR) assay was optimized for the diagnosis of SARS-CoV-2
In the COVID-19 negative specimen, the internal control gene (RP) was the only gene amplified with a sigmoidal amplification curve (Supplementary Fig. S1b)

Summary

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the subfamily Orthocoronavirinae of the family Coronaviridae of the order Nidovirales. The sensitivity of serological tests is limited in the acute stage of infection Another drawback is the possibility of similar antibody responses to the viruses in the same or close families and the possibility of cross-reactivity. According to Fang et al.[12], RT-PCR was able to detect only 71% (36/51) of SARS-CoV-2 infections This may be due to the low sensitivity of the test, low patient viral load, or inappropriate clinical sampling[12]. A multiplex real time RT-PCR (rRT-PCR) assay was designed and evaluated for the diagnosis of SARS-CoV-2 including the most recent variant of concerns (VOC). The developed assay simultaneously detects viral N, RdRP and human RP genes in the same rRT-PCR reaction. The clinical performance of the test was screened with RNA samples from SARS-CoV-2 positive patients

Methods

Results

Discussion

Conclusion