| Bovine parvovirus infection is a contagious disease of cattle caused by bovine parvovirus (BPV). BPV infection mainly causes diseases of the gastrointestinal and respiratory tracts, fetal infection and reproductive disorders. BPV was first isolated in1961by Abinanti and Warfield from the gastrointestinal tract of calves with diarrhea. Due to its ability to hemadsorb erythrocytes on infected bovine embryonic kidney cell cultures, this first isolate was named HADEN (hemadsorbing enteric) virus of calves. Subsequently, the virus isolated from cattle of the outbreak of BPV infection, identification and serology survey the results showed that BPV infection have a higher prevalence in some herds, also serologic survey reported the disease is a worldwide epidemic. At present, bovine parvovirus infection study is relatively small, whether our cattle have bovine parvovirus infection, and how degree of the danger is still lack of clear background information. In the practice, bovine parvovirus infection lack of detection of antigen and antibody reagents were also restricted our detection of the disease and pathogen. So research and development of a sensitivity and specificity detection kit to the etiology and serological test reagent for bovine parvovirus infection is particularly important.At first, a loop-mediated isothermal amplification (LAMP) assay was developed for detection of bovine parvovirus (BPV) DNA in this study. According to the sequences of bovine parvovirus published on GenBanK using biology software alignment to select the highly conserved sequence in the VP2region as the target gene sequence. A set of primers were designed to recognize six distinct regions on the target DNA based on the target gene sequence. The total DNA extracted from BPV primary cell culture was used as a template for LAMP conservative parameters of amplification. An evaluation under different concentrations of MgSO4, betaine, the ratio of outer and inner primers, the amplification temperature and the reaction timewas carried out to optimize the LAMP reaction conditions. The results showed that the optimized LAMP reaction conditions were8mM Mg2+,1.2mM betaine,0.2μM F3and B3primers,1.6μM FIP and BIP primers, the ratio of outer and inner primers8:1, and an incubation at63℃for1h. The detection limit of the LAMP assay was9copies of BPV-DNA and was100times more sensitive than conventional PCR. A ladder pattern of bands after gel electrophoresis was observed for only BPV isolates and showed that the developed BPV LAMP assay method was highly specific without any cross-reactivity with other related viruses(BRV、BVDV、BHV、GPV、PPV). After amplification the products were detected either by observing a adder pattern following gel electrophoresis, observation of turbidity, or a color change with the addition of SYBR Green I to the reaction tube. The LAMP assay was further evaluated using59field samples and the results were comparable to conventional PCR. The results showed that the two methods in line with rate of94.9%. Consequently, the LAMP assay is a sensitive, specific, reproducible detection method; it can provide a useful technique suitable for specific detection of clinical BPV infection.To identify the antigenicity in different VP2regions of bovine parvovirus-1(BPV-1), the VP2gene of BPV-1VR767strain was amplified by PCR and sequenced. Three immunodominant regions of BPV VP2-1(aa34-aa263), VP2-2(aa300-aa451) and VP2-3(aa484-aa633) were analyzed by bio informatics softwares and subcloned into pET28a for expressions in E.coli, which were30.2ku,22.9ku and22.7ku, respectively, identified by western blot. Furthermore, the mice were immunized with the truncated recombinant proteins to prepare the antisera, respectively Western blot and indirect ELISA analysis showed that three truncated proteins specifically reacted with antiserum against BPV. In addition, all of the three proteins were able to induce antibody responses in mice and VP2-3induced the highest antibody titers.An indirect ELISA was developed to detect antibody against BPV using recombinant VP2-3protein of bovine parvovirus. The optimum reaction conditions, including20μg/ml of the VP2-3protein antigen for coating, testing serum dilution at1:40, the best blocking solution of2.5%skim milk, the optimal dilution of HRP-labeled rabbit anti-bovine IgG was1:5000, the optimal OPD-H202color for10min. Criteria for the OD490nm≥0.185sentenced for antibodies-positive,otherwise sentenced for the antibody-negative. There is no any cross reaction between the antigen and the positive serum of BVDV, BHV, BPI3and B.abortus. The method has good specificity and reproducibility.Comparison to the HI Test, the agreement rate of ELISA is91.4%. The678serum samples from Yichun of Heilongjiang province were detected by this assay. The positive rate is52.1%. The result is that this ELISA could be used for diagnosis and epidemiological investigation for BPV to provide an effective means of technology. |
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