| Infectious bovine rhinotracheitis(IBR)and Bovine viral diarrhea(BVD) are two mainly infection illness that lead to cow propagation and cause huge economic losses. The two illness should be inspected to import and exit bovine base on the inspection order. Bovine semen can carry these virus, that can infect other health bovine through inseminating. Virus isolation is the main detection method to IBRV and BVDV in bovine semen, but the method has some difficulty such as lower sensitivy, operation complex and cost a long time, and so on. It is important to know the relation of semen carrying virus and serum antibody in semen produce center, that decide wether the bovine is valuble. There were not report about IBRV and BDVD detection used molocole biology technology. Based on the inspection and quarantine of entry and exit, following results were achieved in the study:1. 684 bovine serum samples antibody against IBRV coming Xinjiang 17 different regions were examined used Institute pourquiers company ELISA kit, 370 serum samples were conformed positive. The postive rate was between 5% and 97.6%, the mean postive rate was 54.1%. There were four farms that had a clear record about abortion in the past three years, in of them, IBRV serum antibody postive rate were 100% in three farms and one farm was 12.5%, the mean postive rate was 90.5%. The results showed that IBRV had an extensive exist in Xinjiang province. The infection rate had no a clear region distribution.2. According to the gB gene sequence of Bovine Herpesvirus 1(BHV1), primers and fluorescent probe were designed to amplify the nucleotide fragment containing the real-time PCR amplification region. An Internal Amplification Contro(lIAC)template of real-time PCR was achieved by amplifying with bridge-building PCR method, and the real-time PCR detection system with IAC was established. The detective limit was about 10 copies/PCR reaction. The real-time PCR with IAC was used directly detection IBRV in raw semen and extended semen samples.The detective limit was about 0.02 TCID50. Compared with OIE reference method, the real-time PCR was 40 folds more sensitive,and that was 500 folds and 50 more sensitivity compared virus isolation and PCR assay respectively. 120 bovine fresh semen samples and 40 extended semen samples and 39 nose swabs were detected with the real-time PCR with IAC,27 samples were positive. These results were almost coincident with real-time PCR without IAC. What is more, the IAC in the real-time PCR system can indicate and proofread false negative results. Serum antibody against IBRV of 10 bulls collected regularly were detected at the same time, and the results showed the bull can not be confirmed whether the semen carrying virus through serum antibody, vice verse. Nose swabs carrying virus was intermittent.3. 875 bovine serum samples antibody against BVDV coming Xinjiang different region were detected used Idexx company indirect ELISA kit, 759 serum samples were conformed positive. The postive rate was between 55.0% and 100%, and the mean postive rate was 86.7%. The results showed that the positive rate was clearly heigher to aborted bovine and the infection had no a clear different to different age bovine. IBRV had an extensive exist in Xinjiang province and is becoming more and more. Compared with VN method, the ELISA was convenient for operation, and was sensitive and specific to BVDV antibody detection. The agreement was 80.0% to VN.4. 6 bovine serum were conformed postive by Idexx company antigen-capture ELISA kit from 160 bovine serum and 20 anus swabs were inoculated by MDBK cells. The 6 serum and 2 anus swabs caused cell pathological. TCID50 of the 8 virus strains were 4.38×103 to 5.82×106. The Serum neutralization titer was 1:23 to1:26. The homology of the 8 islolated strains were 82.0 % to 94.8% and 81.0% to 82.0% compared with NADL strain and KE9 strain respectively. The results showed that the gene type were samely between the 8 isolated virus and NADL strain.5. The fluorescent quantitative RT-PCR (FQ-RT-PCR) method was established for direct detection BVDV in raw semen and extended semen samples. The level of sensitivity was about 0.0125TCID50 for semen samples. The FQ-RT-PCR assay in this study was 200 to 2000 times more sensitive than virus isolation, and that was 100 folds more sensitive compared with conventional RT-PCR. 120 bovine raw semen samples and 40 extended semen samples were collected within 6 months from a bull farm and detected with the FQ-RT-PCR. All the samples were negative. In of them, serum antibodies against BVDV of 10 bulls collected regularly were detected with VN at the same time. The results showed the bull can not be confirmed whether the semen carrying virus through serum antibodies and the antibody can maintain at least half a year.6. According to the published N. caninum Nc-5 gene and BHV-1 gB gene and BVDV 5'UTR, primers and probes were designed. After PCR amplication and gene clone, recombiant plasmids were achieved and mult-PCR method was developed of the three illness. The denaturated PCR products marked fluorescent dye were hybridized to the gene chip, after the optimization of reaction condition, the gene chip diagnostic technique detecting IBRV and BVDV and N. caninum. was established. The sensitivity of the gene chip method was 103 copies each reaction to recombiant plasmids of IBRV and BVDV and N. canimum. The sensitivity to BVDV and IBRV in bovine semen was 0.1TCID50. The application of detection proved the method was repecific and reliable.7. Suspension array of detection IBRV and BVDV and N. caninum was establishe in the study. The single test and mult-test of the suspension array both showed the method was repecific and reliable, and the sensitivity was 102 copies each reaction to recombiant plasmids of BVDV, and that was 103 copies each reaction to N. canimum. and IBRV. The sensitivity to BVDV and IBRV in bovine semen was 0.01TCID50.That was 100 folds more sensivity compared to PCR. The application of detection proved the method was viable.Some samples were detected with virus isolation, PCR, real-time PCR, gene chip and suspension array, and so on. The results showed the real-time PCR had the highest sensitivity compared with gene chip and suspension array, but gene chip and suspension array can detection illness at the same time. There were some report about animal illness detection used real-time PCR, gene chip and suspension array by now. But without study on these different method as a whole, and no a further study report on quality control of animal illness detection. The built of technology platform of DNA virus, RNA virus and parasites illness detection was contributed to more animal illness study in the future. The analysis of semen carrying virus and serum antibody was finished at the same time. All these were helpful to constitution of inspection and quantine policy and to choice of detection method and to afford necessary technology store to animal entry and exit inspecction and quarantine.
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