key: cord-0879003-pli1ugoa
authors: Wang, Yong; Sun, Jianfei; Guo, Xu; Zhang, Da; Cui, Yongqiu; Li, Wei; Liu, Guangqing; Li, Yongdong; Jiang, Shudong
title: TaqMan-based real-time polymerase chain reaction assay for specific detection of bocavirus-1 in domestic cats
date: 2020-08-08
journal: Mol Cell Probes
DOI: 10.1016/j.mcp.2020.101647
sha: 78cbb53a2ad7bc2a4b00eff7a8761407d52a1893
doc_id: 879003
cord_uid: pli1ugoa

Feline bocavirus-1 (FBoV-1) was first discovered in Hong Kong in 2012, and studies have indicated that the virus may cause feline hemorrhagic enteritis. Currently, there is a lack of an effective and quantitative method for FBoV-1 detection. In this study, a TaqMan-based quantitative real-time PCR (qPCR) for FBoV-1 detection was established. Primers and probes were designed to target the conserved region of the FBoV-1 NS1 gene. The sensitivity analysis indicated that the minimum detection limit was 4.57 × 10(1) copies/μL. The specificity test revealed no cross-reaction with seven other common feline viruses, including the same species—FBoV-2 and FBoV-3. The sensitivity of this method was 100 times higher than that of conventional PCR (cPCR). The established method showed good repeatability, with the intra-assay and inter-assay coefficients of variation of 0.18%–1.00% and 0.27%–0.45%, respectively. Furthermore, the analysis of feline feces revealed that the detection rate by qPCR was 7.0% (9/128), whereas that by cPCR was 4.7% (6/128). In conclusion, the established qPCR assay can quantitatively detect FBoV-1 with a high sensitivity, high specificity, and good reproducibility, making it a promising technique for the clinical detection of and basic and epidemiological research on FBoV-1.

Bocavirus (BoV) belongs to the subfamily Parvovirinae and is a non-enveloped, 46 linear, single strand DNA (ssDNA) virus with a genome of 5.4 kb [1] [2] [3] [4] [5] The subsequent reports indicated that FBoV-1 could be easily detected in cats with 55 diarrhea compared with healthy cats [2] . Notably, FBoV-1 has been closely related to Establishing an efficient FBoV-1 clinical detection method can provide a 60 powerful tool for the diagnosis of BoV infection in cats with and without diarrhea.

However, currently, only conventional PCR (cPCR) has been established for FBoV 62 detection. Thus, there is a need to develop a more rapid, sensitive, and specific 63 method to detect FBoV-1. TaqMan assay has the characteristics of high sensitivity and 64 specificity, and it can be used for quantitative analysis. Indeed, TaqMan assay is now becoming the first choice for pathogen detection in clinical samples [11, 12] . In this 66 study, we aimed to establish a sensitive and specific TaqMan-based quantitative Table 1 . The optimized qPCR assay was used to detect 10-fold serially diluted 122 recombinant plasmids, and the minimum detection limit of the method was analyzed 123 using a standard curve. The forward and reverse primers used for the qPCR were used 124 for the cPCR. Hence, cPCR was used to detect the template with the same 125 concentration gradient, and agarose gel electrophoresis was carried out to observe the 126 amplified product. Finally, the sensitivity of qPCR and cPCR was compared.

To evaluate the specificity of the qPCR established in this study, the assay was and FBoV-3) and the negative control (RNase-free H 2 O) tested negative (Fig 3) . 158 The results showed that the intra-assay and inter-assay SDs ranged from 0.05 to 159 0.14 and 0.02 to 0.09, respectively. Furthermore, the intra-assay and inter-assay 160 coefficients of variation (CV) ranged from 0.18% to 1.00% and 0.08% to 0.45%, 161 respectively (Table 2) . 163 The qPCR method established in this study and the cPCR method reported 164 previously were used to detect the 128 collected samples. The results showed that the 165 detection rate using the qPCR method was 7.0% (9/128) and that using the cPCR 166 method was 4.7% (6/128). Furthermore, the positive rate of detection of samples from 167 cats with diarrhea using the qPCR method was 10.2% (9/88); FBoV-1 was not 168 detected in cats without diarrhea. All test samples positively detected by cPCR could 169 also be detected by qPCR (Table 3) J o u r n a l P r e -p r o o f Table 2 The repeatability of the established qPCR. 

Characterization of novel canine bocaviruses and their 240 association with respiratory disease

Detection and genetic characterization of feline bocavirus in Northeast China

The 246 family Parvoviridae