| Rabbit hemorrhagic disease (RHD) is a highly contagious disease against the rabbits healthy development, and it caused by the rabbit hemorrhagic disease virus (RHDV). This disease mostly kills the young rabbits over 2 months, and has a very high morbidity and mortality. So it reduces the economic benefits of the rabbit farming industry. Since 2010, the European area infected rabbit hemorrhagic disease mutant strain RHDV2, which caused the death of young and vaccinated rabbits. This situation drew wide attention in the rabbit industry. In view of mutant circumstances and new emerging epidemiological trends of RHDV,we analyzed the capsid protein gene VP60 of RHDV and RHDV2 to establish the SYBR Green I real-time RT-PCR detection method respectively. This study provided scientific and technical materials for quick detection diagnosis and molecular epidemiology of RHD.According to the conserved region of RHDV2 VP60 gene that published on NCBI, we designed 6 pairs of primers to synthesize target gene. Then purified the target gene and constructed the recombinant plasmid pMD19-T-RHDV2. A pair of degenerate primer was designed in the region of target gene. The positive plasmid was used as templates for PCR amplification to establish the SYBR Green I real-time RT-PCR detection method of RHDV2.Then optimized the reaction conditions, and determined the sensitivity, specificity and reproducibility of this methods. At the same time, this method was used to detect the artificial infection samples and clinical samples. The results showed that the SYBR Green I real-time RT-PCR method had a high sensitivity, specificity and reproducibility. And it could detect pMD19-T-RHDV2 with a limited detection of 68 copies. It could specifically detect RHDV2 and there was no amplification of RHDV, Pasteurella multocida, Salmonella, Escherichina coli and healthy organs from rabbits. The variation coefficients of Ct values that obtained by the amplification of positive plasmids in the group were less than 0.8%, and the detection results of RHDV from artificially infected samples and clinical samples were negative.By comparing and analyzing RHDV VP60 gene sequences on NCBI, a pair of specific primer in the conserved region was designed, and the RNA of RHDV from positive samples was extracted and reverse transcribed. The production of reverse transcription was served as template for PCR amplification to obtain a 979bp target fragment. The target gene was purified and cloned into pMD19-T vector, then transferred into the E.coli DH5a to construct the recombinant plasmid pMD19-T-RHDV, which was served as template to establish the standard curve. Meanwhile, a pair of degenerate primers amplifying 165 bp target fragment in the amplified region was designed for fluorescence quantitative RT-PCR amplification. By optimizing the reaction conditions and procedures, and authenticated the sensitivity, specificity and reproducibility assays to establish the SYBR Green I real-time RT-PCR detection method of RHDV. Then using the established method to detect and analysis the virus content in different organs of artificially infected samples(7 rabbits). At the same time, using the RT-PCR method that had been established previously to detect each organ. The results showed that the SYBR Green I real-time RT-PCR can detect pMD19-T-RHDV2 with a limited detection of 81 copies. It was more sensitive than RT-PCR. This method could specifically detect RHDV, however, there was no amplification of pMD19-T-RHDV2, Pasteurella multocida, Salmonella, Escherichina coli and healthy organs from rabbits. The interclass variation coefficients of Ct values obtained by the amplification of positive plasmids were less than 1.12%, which indicated that the method had high sensitivity, specificity and reproducibility. The amplification results of each organ of the artificial infected rabbits showed that the spleen is the most viral organ, and the positive rate by the real-time PCR is 100%. Otherwise, the virus content of each sample can be calculated to achieve the effect of accurate quantitative according to the Ct values. The detection results of RT-PCR showed that the electrophoretic strip of spleen is the most clear and viral, basically consistent with the results of real-time RT-PCR. |
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