Development of multiplex real-time PCR for simultaneous detection of SARS-CoV-2, CCoV, and FIPV

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• One Health

Background:

• Coronaviruses like SARS-CoV-2, CCoV, and FIPV pose interspecies transmission risks.
• Coexistence of humans, dogs, and cats creates complex environments for viral spread.
• Threats to both human and animal health necessitate effective diagnostic tools.

Purpose of the Study:

• To develop a novel multiplex real-time PCR assay.
• To enable simultaneous detection and differentiation of SARS-CoV-2, CCoV, and FIPV.
• To enhance diagnostic efficiency and reduce costs for coronavirus surveillance.

Main Methods:

• Designed specific primers and TaqMan fluorescent probes for SARS-CoV-2, CCoV, and FIPV.
• Utilized a multiplex TaqMan-probe-based real-time PCR approach.
• Validated the assay using plasmid and clinical samples.

Main Results:

• Achieved high sensitivity with detection limits as low as 9.25 copies/μL for FIPV.
• Demonstrated high amplification efficiency (91%-116%) and R(2) values (≥0.95).
• Showed strong agreement with conventional methods in analyzing 91 samples.

Conclusions:

• The multiplex assay enhances testing efficiency and reduces costs.
• Provides a valuable tool for tracking prevalence and distribution of co-infected animals.
• Contributes to the advancement of animal and public health through improved diagnostics.