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

Area of Science:

• Virology
• Molecular Diagnostics
• Public Health

Background:

• Acute respiratory tract infections (ARTIs) pose a significant global health challenge, particularly for young children and the elderly.
• Viral pathogens are the primary cause of ARTIs, necessitating accurate and rapid diagnostic methods for effective patient management.

Purpose of the Study:

• To develop and validate a sensitive and specific multiplex RT-qPCR assay for the simultaneous detection of 18 common respiratory viruses.
• To evaluate the performance of the in-house assay against commercial kits using clinical samples.

Main Methods:

• Development of a multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay.
• Assay validation for sensitivity, specificity, and linear correlation using defined viral loads.
• Performance evaluation with 628 clinical samples, comparing results to commercial diagnostic kits.

Main Results:

• The in-house multiplex RT-qPCR assay demonstrated high sensitivity (LOD: 5-75 copies/25 μL) and excellent specificity with no cross-reactivity.
• Clinical evaluation showed 99.81% sensitivity, 100% specificity, and 99.84% overall consistency for six key viruses (IAV, IBV, RSV, HRV, HAdV, SARS-CoV-2).
• High prevalence of influenza A virus (IAV), human adenovirus (HAdV), and human rhinovirus (HRV) was observed, with a notable potential for HRV co-infection.

Conclusions:

• The developed multiplex RT-qPCR assay is a sensitive, specific, and cost-effective tool for detecting 18 common respiratory viruses.
• This assay can significantly aid in the clinical management and epidemiological surveillance of ARTIs.
• Findings highlight the prevalence of specific viruses and co-infections in the Taizhou region, informing public health strategies.