Quantitative multiplex detection of 9 feline respiratory pathogens with enhanced sensitivity: a cost-effective integrated platform combining RT-PCR/PCR and capillary electrophoresis

Pathogen infections and coinfections are critical determinants of feline upper respiratory tract disease; although the etiologic agents are diverse, their clinical manifestations often overlap. Established detection panels for feline upper respiratory tract infections (FURTIs) primarily focus on core pathogens—feline calicivirus (FCV), felid alphaherpesvirus 1 (FeAHV1), Mycoplasmopsis felis (MF), Chlamydia felis (CF), Bordetella bronchiseptica (BB), and influenza A virus (IAV)—and frequently do not incorporate emerging zoonotic agents (e.g., SARS-CoV-2) and overlooked streptococcal species ( Streptococcus equi subsp. zooepidemicus [SEZ] and S. canis [SC]) in a multiplex assay. Furthermore, simultaneous quantification remains technically challenging. We developed an integrated multiplex reverse-transcription PCR (RT-PCR)/PCR–capillary electrophoresis (CE) platform for simultaneous quantitative detection of 9 feline respiratory pathogens (FCV, FeAHV1, MF, CF, BB, IAV, SARS-CoV-2, SEZ, SC). Requiring only a standard thermal cycler and a basic CE instrument, our platform is highly accessible to resource-limited laboratories, offering high specificity and a 1.6-h turnaround time. With a limit of detection of 1 copy/µL across all targets, our multiplex assay had exceptional sensitivity, surpassing conventional singleplex RT-PCR/PCR on both agarose gel and real-time PCR platforms. Notably, we achieved a 2.2-fold higher detection rate than the conventional gold standard (singleplex RT-PCR/PCR with agarose gel). This enhanced sensitivity enabled us to identify even low-pathogen–load samples, often missed by conventional assays. When used with clinical specimens, the platform offered comprehensive coverage of major and emerging zoonotic FURTI pathogens, confirming its potential as a cost-effective, high-efficiency tool for differential diagnosis, targeted therapy, and epidemiologic surveillance.