Development and validation of multiplex one-step qPCR/RT-qPCR assays for simultaneous detection of SARS-CoV-2 and pathogens associated with feline respiratory disease complex

Feline respiratory disease complex (FRDC) is caused by a wide range of viral and bacterial pathogens. Both Influenza A virus (IAV) and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) also induce respiratory diseases in cats. Two one-step multiplex qPCR/RT-qPCR assays were developed and validated: FRA₁ (Feline respiratory assay 1) for the detection of four viral targets and FRA₂ for the detection of three bacteria associated with FRDC. Both multiplex assays demonstrated high specificity, efficiency (93. 51%–107. 8%), linearity (> 0. 998), analytical sensitivity (≤ 15 genome copies/μl), repeatability (coefficient of variation CV < 5%), and reproducibility (CV < 6%). Among the 63 clinical specimens collected from FRDC-suspected cats, 92. 1% were positive for at least one pathogen and co-infection was detected in 57. 1% of samples. Mycoplasma felis (61. 9%) was the most found pathogen, followed by feline herpesvirus-1 (30. 2%), Chlamydia felis (28. 7%) and feline calicivirus (27. 0%). SARS-CoV-2 was detected in two specimens. In summary, this new panel of qPCR/RT-qPCR assays constitutes a useful and reliable tool for the rapid detection of SARS-CoV-2 and viral and bacterial pathogens associated with FRDC in cats.