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

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.

Influenza A, Influenza B, and SARS-CoV-2 Similarities and Differences – A Focus on Diagnosis

“…False-negative rates can be lowered by considering clinical presentation and associating antibody testing ( Teymouri et al, 2021 ). Additionally, to limit false-negative results, particularly in the setting of low viral loads, current guidelines recommend the simultaneous detection of at least two target SARS-CoV-2 genes ( World Health Organization [WHO], 2020 ; Tombuloglu et al, 2022 ). Virus mutation rates also influence false-negative rates.…”

A comparison of conventional and advanced electroanalytical methods to detect SARS-CoV-2 virus: A concise review

“…3 . RT-PCR is one of the nucleic acid amplification strategy that converts RNA into complementary deoxyribonucleic acids (cDNA), which is then rigorously amplified for detection ( Tombuloglu et al, 2021 , 2022 ; Gerard et al, 1997 ). In the collected NPS samples, RT-PCR identifies the presence of viral nucleic acids.…”

“…The research suggests that further work is needed to tackle amplification and sensitivity difficulties when it comes to identifying SARS-CoV-2 using conventional methods. PCR offers a number of advantages and distinguishing characteristics that make it a viable diagnostic tool ( Tombuloglu et al, 2021 , 2022 ). For example, during the early phases of infection, RT-PCR can be utilized to detect the viral genome sequence.…”

COVID-19 diagnostic methods in developing countries

“…As a result, and using RNA-dependent DNA polymerase, cDNA copies were produced. In RT-PCR, this amplification is monitored using various fluorescence dyes or DNA probes labeled with fluorescence molecules that target a specific part of the genome (Tombuloglu et al 2022 ).…”