Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of four feline diarrhea-associated viruses

Abstract

Since their recent discovery, the prevalence of novel feline enteric viruses, including feline bocavirus 1 (FBoV-1), feline astrovirus (FeAstV), and feline kobuvirus (FeKoV), has been reported in China. Co-infections of these viruses with feline parvovirus (FPV) are common causes of diarrhea in cats. Viral co-infections are difficult to identify because of their non-specific clinical signs. To detect and identify these viruses, a quick and specific pathogen-testing approach is required. Here, we establish a real-time PCR (qPCR) based on multiple TaqMan probes for the simultaneous detection of FBoV-1, FeAstV, FeKoV, and FPV. Specific primers and TaqMan fluorescent probes were designed to ensure specificity. The results showed that the detection limit of single qPCR was up to 10 copies, and the detection limit of multiplex qPCR was up to 100 copies, with correlation coefficients >0.995 in all cases. Clinical sample detection revealed a 25.19% (34/135) total rate of co-infection among the viruses and a 1.48% (2/135) quadruple infection rate. Thus, this multiplex qPCR approach can serve as a quick, sensitive, and specific diagnostic tool for FBoV-1, FeAstV, FeKoV, and FPV identification, and it may be utilized for routine surveillance of these emerging and reemerging feline enteric viruses.

Multiplex One-Step RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Enteric Viruses of Dogs and Cats

“…Moreover, in this study, the LOD 95 for CPV and CECoV showed a one log 10 improvement compared to previously published multiplex qPCR assays (LOD 95 was 1 × 10 2 copies/μL) [ 44 ], and a three log 10 improvement compared to a multiplex RT-PCR assay designed for detecting CPV, CECoV, and CAdV-1 [ 67 ]. Similarly, our FEA_1 assay displayed a reduction of more than one log 10 in detecting FPV when compared to another multiplex RT-qPCR assay designed for detecting FPV, feline bocavirus 1, feline astrovirus, and feline kobuvirus [ 68 ]. Furthermore, all the previous assays were conducted in two separate steps (reverse-transcription and subsequent qPCR amplification), while our newly developed one-step assays combined both the reverse-transcription and qPCR amplification steps.…”