Development of a TaqMan probe - based PCR for the detection of porcine bocavirus group 3 and identification of a novel genotype strain

For specific detection of porcine bocavirus (PBoV) group 3 (G3), we developed a TaqMan probe-based real-time polymerase chain reaction (PCR) targeting the NS1 gene. The correlation coefficient and amplification efficiency of the PCR were 0.994 and 102.9%, respectively. There were no cross-reactions of the PCR with the genomes of classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV), and the detection limit of the PCR was 100 copies per reaction. The coefficients of variation of the inter-assay and intra-assay of the PCR were 1.38-2.71% and 0.23-0.67%, respectively. A total of 719 field samples collected from Xinjiang, Gansu, Qinghai, Ningxia, and Shaanxi Provinces were detected via the developed PCR, and the positive rates were 80.82%, 44.51%, 97.50%, 98.94%, and 93.60%, respectively. In addition, coinfections of PBoV G3 with PEDV/TGEV/PDCoV/PCV2 were determined, and dual infections, triple infections, quadruple infections, and quintuple infections were observed. Furthermore, the complete genome sequences of 10 positive samples (two per province) randomly selected from five provinces were determined, and phylogenetic analysis placed all 10 within PBoV G3, but one strain formed a distinct branch, indicating it was a novel strain in PBoV G3. The established PCR provides a reliable tool for PBoV G3 detection and facilitates the understanding of its distribution and prevalence. IMPORTANCE: Porcine bocavirus (PBoV) poses a threat to swine health and has potential public health significance. Owing to the high nucleotide diversity of PBoV, existing detection methods may yield false-negative results. A reliable detection method is needed to monitor its prevalence. Given that porcine bocavirus group 3 (PBoV G3) is the predominant group circulating in Chinese pig populations, we established a rapid and sensitive TaqMan probe-based polymerase chain reaction (PCR) for detection of PBoV G3. We detected 719 field samples collected from northwest China by the PCR and analyzed coinfections of PBoV G3 with porcine epidemic diarrhea virus/transmissible gastroenteritis virus/porcine deltacoronavirus PCV2. The results showed that the positive rate of PBoV G3 was high, and dual infections, triple infections, quadruple infections, and quintuple infections were present. Furthermore, a novel genotype was identified in PBoV G3. Taken together, the developed PCR facilitates the prevalence investigation of PBoV G3. The results enrich the data on the distribution and prevalence of PBoV G3 in China.