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. 2022 Nov 3:9:1005759.
doi: 10.3389/fvets.2022.1005759. eCollection 2022.

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

Junwei Zou  1 , Ju Yu  1 , Yuanyuan Mu  1 , Xiangyu Xie  1 , Run Wang  1 , Haiqiang Wu  1 , Xuan Liu  1 , Fazhi Xu  1 , Juhua Wang  1 , Yong Wang  1
Affiliations

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

Junwei Zou et al. Front Vet Sci. .

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.

Keywords: FBoV-1; FPV; FeAstV; FeKoV; TaqMan-based multiplex qPCR; co-infection; detection.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
(A–D) The amplification curves (top, X-axis: Cycle, Y-axis: Fluorescence) and standard curve (bottom, X-axis: Log Quantity, Y-axis: Cq) for the single qPCR assay of individual viruses. (A) Feline parvovirus (FPV) plasmids from 108 to 101 copies/μL. (B) Feline bocavirus 1 (FBoV-1) plasmids from 108 to 101 copies/μL. (C) Feline kobuvirus (FeKoV) plasmids from 108 to 101 copies/μL. (D) Feline astrovirus (FeAstV) plasmids from 108 to 101 copies/μL.
Figure 2
Figure 2
Development of a multiplex qPCR detection method. (A) The amplification curves (top, X-axis: Cycle, Y-axis: Fluorescence) and standard curve (bottom, X-axis: Log Quantity, Y-axis: Cq) for a multiplex qPCR assay of feline parvovirus (FPV), feline bocavirus 1 (FBoV-1), feline kobuvirus (FeKoV) and feline astrovirus (FeAstV). Each virus plasmid is from 108 to 102 copies/μL. (B) Standard curve formulae in the multiplex qPCR for FPV, FBoV-1, FeKoV, and FeAstV.
Figure 3
Figure 3
The specificity of the multiplex qPCR. Feline bocavirus 1 (FBoV-1), feline parvovirus (FPV), feline kobuvirus (FeKoV), and feline astrovirus (FeAstV) showed specific amplification curves in the multiplex qPCR assay. Other samples include feline bocavirus 2 (FBoV-2), FBoV-3, feline coronavirus (FCoV), feline chaphamaparvovirus (FeChPV), and nuclease-free water.
Figure 4
Figure 4
Co-infection analysis of detected feline viruses. Total 135 clinical feline fecal samples collected from 2018 and 2021 were analyzed by the multiplex real-time qPCR assay. A total of 92 samples were detected as positive. The Venn diagram shows the number of samples infected by either single virus or multiple viruses.
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