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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: FRA_1 (Feline respiratory assay 1) for the detection of four viral targets and FRA_2 for the detection of three bacteria associated with FRDC. Both multiplex assays demonstrated high specificity, efficiency (93.51%\u2013107.8%), linearity (&gt; 0.998), analytical sensitivity (\u2264 15 genome copies\/\u03bcl), repeatability (coefficient of variation [CV] &lt; 5%), and reproducibility (CV &lt; 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. <jats:italic>Mycoplasma felis<\/jats:italic> (61.9%) was the most found pathogen, followed by feline herpesvirus-1 (30.2%), <jats:italic>Chlamydia felis<\/jats:italic> (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.<\/jats:p>","DOI":"10.1371\/journal.pone.0297796","type":"journal-article","created":{"date-parts":[[2024,3,22]],"date-time":"2024-03-22T17:22:58Z","timestamp":1711128178000},"page":"e0297796","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":13,"title":["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"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7402-5989","authenticated-orcid":true,"given":"C\u00f4me J.","family":"Thieulent","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariano","family":"Carossino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"Peak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wendy","family":"Wolfson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0609-678X","authenticated-orcid":true,"given":"Udeni B. 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