| Canine parvovirus (CPV) has been considered a highly contagious, fatal infectiousdisease in domestic and wild canids diseases, characterised by severe vomiting, bloodydiarrhea, watery diarrhea with the stench, myocarditis, severe dehydration and whiteblood cell decreasing significantly. Our study is to establish a rapid PCR method fordetecting samples of suspicious parvovirus infection. Meanwhile, to clone CPV-VP2gene and its prokaryotic expression and renaturation, and establish indirect ELISA ofCPV antibody detection, initially applie to CPV sera antibody detection of dogs.1. Primers were designed according to the sequences of CPV gene in retrievedfrom GenBank, we utilized samples of suspected CPV infection for PCR amplification,amplified products were analyzed by sequencing, and the homology was99.8%~100%compared to other CPV nucleotide sequence in NCBI. The results showed it was CPVpositive. Twenty-eight samples of suspected CPV infections were collected from JiangxiAgricultural University veterinary hospital, the positive rate was89.28%(25/28) byPCR, and the positive rate was71.42%(20/28) by colloidal gold assay. It was showedthat the sensitivity of PCR was significantly higher than the colloidal gold assay.2. The primers were designed according to CPV-VP2sequence in retrieved fromGenBank, the length of VP2gene was1755bp amplified by PCR, we inserted it into Tvector and sequenced, the nuclear nucleotide homology was99.0%~99.7%compared toother strains separated from different regions in China, and the amino acid homologywas98.8%~99.8%. The results indicated that Nanchang endemic CPV strains were nosignificant genetic variations.3. The VP2gene was inserted into pET32a vector, and recombinant plasmidpET-32a-VP2was constructed successfully and expressed in BL21. We obtainedrecombinant VP2protein (1.3634mg/mL) through ultrasonic lysis, protein purificationand renaturation. Kunming mice were immunized with it to establish an indirect ELISA,the results showed sera antibody titer of it was higher than1:51200, VP2protein hadgood immunogenicity. We established successfully CPV-VP2protein antibody indirectELISA with VP2protein in7.5mg/mL coating ELISA plates, sera dilution of1:40, HRPsecondary antibody concentration of1:6000, and it can be used to detect the antibodiesof canine CPV induced by infection or vaccination.A total49samples of healthy canine sera stored at Jiangxi Agricatural UnivesityVeterinary hospital were tested by indirect ELISA, results:7sera were negative in thedogs without inoculated CPV vaccine, however, among the42vaccinated dogs,36serawere positive for CPV and6sera were suspicious.Preliminary study indicated that themethod can be applied to the detection of antibodies of CPV. |
