Development and Validation of a Triplex RT-qPCR Assay for Rapid Clinical Diagnosis and Serotyping of Feline...

Feline infectious peritonitis (FIP) is a highly lethal disease caused by feline infectious peritonitis virus (FIPV), which poses significant diagnostic challenges in clinical practice. FIPV is divided into two serotypes (serotype I and serotype II) based on distinct serological responses driven by substantial sequence divergence in the spike (S) protein. Serotype I predominates in Europe and North America, whereas serotype II is more common in Asia. In this study, we developed a triplex reverse transcription quantitative PCR (RT-qPCR) assay for simultaneous detection and serotyping of FIPV. Primers and TaqMan probes were designed to target the conserved nucleocapsid (N) gene and serotype-specific regions within the S gene. After systematic optimization of reaction conditions, the final assay employed an annealing temperature of 64 °C and optimized primer-probe concentrations. The assay exhibited excellent linearity (R² > 0.99 for all targets), with amplification efficiencies ranging from 97.39% to 109.97%. No cross-reactivity was observed with other common feline pathogens, confirming high specificity. The limit of detection was as low as 0.5 copies/µL, and intra-assay repeatability showed coefficients of variation below 2.1%. Clinical validation using 63 feline samples revealed an overall FIPV positivity rate of 21.63%, with serotype II (17.46%) markedly more prevalent than serotype I (3.17%). Collectively, this triplex RT-qPCR assay demonstrates high sensitivity, exceptional specificity, and robust reproducibility, making it a valuable tool for rapid clinical diagnosis through the simultaneous detection of feline coronavirus (FCoV) and serotyping of FIPV.