Development of a multiplex real-time RT-PCR assay for simultaneous detection and differentiation of influenza A, B,...

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Infectious Diseases

Background:

• Influenza viruses (A, B, C, and D) cause significant respiratory disease in various hosts.
• Accurate and rapid detection of multiple influenza strains is crucial for disease management and control.
• Existing diagnostic methods may lack the capacity for simultaneous detection of all influenza types.

Purpose of the Study:

• To develop and validate a multiplex real-time RT-PCR assay for the simultaneous detection of influenza A, B, C, and D viruses (IAV, IBV, ICV, IDV).
• To assess the assay's specificity, sensitivity, and strain coverage for efficient influenza diagnostics.

Main Methods:

• Design of a multiplex real-time RT-PCR assay targeting unique sequences in the matrix gene (IBV, ICV, IAV) and RNA polymerase subunit PB1 (IDV).
• Inclusion of the host 18S rRNA gene as an internal control for assay validity.
• Validation using transcribed RNA, viral isolates, and clinical samples.

Main Results:

• The assay demonstrated high in silico strain coverage: 97.9% for IBV, 99.5% for ICV, and 100% for IDV.
• Specific detection of target influenza viruses without cross-reactivity with other common pathogens.
• Achieved a limit of detection of approximately 30 copies per RNA template (Ct value ~37).

Conclusions:

• The developed multiplex real-time RT-PCR assay is a sensitive and specific tool for simultaneous detection of IAV, IBV, ICV, and IDV.
• This assay can aid in the rapid diagnosis and epidemiological surveillance of influenza.
• The assay's performance suggests its utility in clinical and research settings for comprehensive influenza diagnostics.