# Development of a multiplex real-time RT-PCR assay for simultaneous detection of 18 respiratory viruses

## TL;DR

A new RT-qPCR test detects 18 respiratory viruses accurately and efficiently, helping diagnose and manage acute respiratory infections.

## Contribution

A novel multiplex RT-qPCR assay with high sensitivity and specificity for 18 respiratory viruses is developed and validated.

## Key findings

- The assay has a detection limit of 5–75 copies/25 μL and strong linear correlation (R²≥0.9944).
- The assay showed 99.81% sensitivity and 100% specificity when tested on 628 clinical samples.
- High prevalence of IAV, HAdV, and HRV was observed in Taizhou, China, with frequent HRV co-infections.

## Abstract

Acute respiratory tract infections (ARTIs) are a major global health burden with high morbidity and mortality, mainly affecting children under 5 years old and people over 60 years old. The majority of ARTIs are caused by respiratory viruses, such as influenza virus (IFV), respiratory syncytial virus (RSV), human rhinovirus (HRV), human parainfluenza virus (HPIV), human adenovirus (HAdV), human coronavirus (HCoV), human bocavirus (HBoV) and human metapneumovirus (HMPV). Accurate and sensitive diagnosis of respiratory viruses is crucial for clinical treatment and management of patients with ARTIs. Here, we developed a multiplex RT-qPCR assay for detection of 18 common respiratory viruses. The in-house multiplex RT-qPCR assay possesses high sensitivity (limit of detection [LOD]: 5–75 copies/25 μL) and a good linear correlation between the logarithmic copy number and cycle threshold (Ct value, R²≥0.9944), along with excellent specificity characterized by no cross-reactivity within the 18 viruses and no non-specific amplification for other human pathogens. The in-house assay was assessed using 628 clinical samples and compared with Sansure and Easy Diagnosis kits. The results showed a sensitivity of 99.81%, a specificity of 100%, and an overall consistency rate of 99.84% for six target viruses (IAV, IBV, RSV, HRV, HAdV, and SARS-CoV-2). We also reported high prevalences of IAV, HAdV and HRV in Taizhou, Jiangsu, China, and a high potential of HRV co-infection with other respiratory viruses. The new multiplex RT-qPCR assay offers a sensitive, specific, and cost-effective tool for monitoring 18 common respiratory viruses to mitigate the prevalence of ARTIs.