A multiplex TaqMan real-time PCR for detection and differentiation of four antigenic types of canine parvovirus in China

Sun, Yaru; Cheng, Yuening; Lin, Peng; Yi, Li; Tong, Mingwei; Cao, Zhigang; Wang, Gaili; Li, Shuang; Cheng, Shipeng; Yuan, Wanzhe; Wang, Jianke

Canine parvovirus (CPV) is an important pathogen in domestic dogs, and the original antigenic types CPV-2 and its variants, CPV-2a, 2b and 2c, are prevalent worldwide. A multiplex TaqMan real-time PCR method was developed for the detection and differentiation of four antigenic types of CPV. A set of primers and probes, CPV-305F/CPV-305R and CPV-2-305P (for CPV-2)/CPV-2a-305P (for CPV-2a, 2b and 2c), was able to differentiate CPV-2 and its variants (CPV-2a, 2b and 2c). Another set of primers and probes, CPV-426F/CPV-426R and CPV-2-426P (for CPV-2 and 2a)/CPV-2b-426P (for CPV-2b)/CPV-2c-426P (for CPV-2c), was able to differentiate CPV-2a (2), CPV-2b, and CPV-2c. With these primers and probes, the multiplex TaqMan real-time PCR assay detected effectively and differentiated CPV-2, 2a, 2b and 2c by two separate real-time PCRs. No cross reactivity was observed with canine distemper virus, canine adenovirus, and canine coronavirus. The detection limit of the assay is 10(1) genome copies/μL for CPV-2, CPV-2a, CPV-2b, and 10(2) copies/μL for CPV-2c. The multiplex real-time PCR has 100% agreement with DNA sequencing. We provide a sensitive assay that simultaneously detects and differentiate four antigenic types of CPV and the method was also used for quantification of CPVs viral genome.