The Establishment Of RT-PCR Assay For Detection Of And Its Application In RHDV-Contaminated Classical Swine Fever Vaccine

Vaccines derived from tissures and cells are the two kinds of classical swine fever vaccines offered in China market. Classical Swine Fever Lapnizied Virus Strain were injected into rabbits and the spleens and lymph nodes were collected, frozen dry under vacuum conditions after made into emulsion and added some appropriate stabilizer, forming live classical swine fever tissue vaccine. As the tissue vaccine were produced directly with the tissues of rabbits, while the rabbits may be infected by Rabbit Haemorrhagic Disease Virus (RHDV) and some infection were even difficult to be detected, the live classical swine fever vaccine made from spleens and lymph nodes of rabbits might be contaminated by RHDV in the process of production. At present, the main methods used to detect RHDV were HA,HI, neutralization test, ELISA, molecular biological test, however, these methods were mainly used in the diagnosis of RHD, so it was necessary to develop a detection method for RHDV in live swine fever vaccine in order to confirm the safety and efficacy of live classical swine fever vaccine.The primers were designed with Primer 5.0 according to the VP60 gene sequence of RHDV showed in GenBank and 370bp fragment can be amplified and based which RT-PCR test were developed to detect RHDV. The test results showed that 370bp sequence could be amplified only from RHDV and the minimum quality that can be detected were 1×10-6 dilution. The amplification results with rabbit E. coli.rabbit Pasteurella multocida,LaRV,LCSFV, BVDV, RV or PRRSV as templates were all negative,which proves that the RT-PCR constructed were specific and sensitive.The classical swine fever vaccine (live, rabbit tissue origin) polluted with RHDV were detected by sandwich ELISA and RT-PCR. The detection results of RT-PCR show that the RHDV in the contaminated vaccines can be checked out when diluted by 1:100000, and the control group which has no vaccines can be detected when diluted by 1:1000000. The results of sandwich ELISA show that the maximum dilution of the polluted vaccine which RHDV can be checked out was 1:10, however the maximum dilution of the control group was 1:100. It shows that RT-PCR was more sensitive in the detection of RHDV compared with sandwich ELISA.Fifteen batches of live classical swine fever vaccine were detected with ELISA and RT-PCR. The positive rate of RT-PCR was 3/15, while the positive rate of sandwich ELISA was 2/15, which shows that parts of classical swine fever vaccines in China market were contaminated by RHDV, and verifing that RT-PCR established in this study has a better sensitivity than that in sandwich ELISA.The standard operation procedure and quality standard of RT-PCR used to detect RHDV contamination in swine fever vaccine(live,rabbit tissue origin) were developed according to the study, which supplies a substitute method to detect exogenous Rabbit Haemorrhagic Disease Virus(RHDV) contamination in the classical swine fever vaccine.