关键词: 兔病毒性出血症病毒（RHD）, VP60, SYBR Green Ⅰ, 实时荧光定量PCR

Abstract:  Rabbit hemorrhagic disease is a highly contagious disease which causes serious damage to the healthy development of rabbit keeping. In order to establish a quick， efficient and sensitive detection method， a pair of specific primers were designed for PCR amplification to obtain 979 bp sequence of rabbit hemorrhagic disease virus（RHDV） VP60 gene. Then cloned it into pMD19T vector to construct recombinant plasmid named pMD19TVP60， which was served as template to establish the standard curve. Meanwhile， a pair of degenerate primer was designed in the amplification region to establish the SYBR Green Ⅰ realtime PCR detection method， and the reaction condition was optimized， and the sensitivity， specificity and reproducibility were tested. The results showed that the SYBR Green Ⅰ realtime PCR could specifically detect RHDV that the limited detection content was 8.18×101 copies and no amplification of pGM19TEBHSV， Pasteurella multocida， Salmonella， Escherichina coli and healthy organs from rabbits. At the same time， this method was used to detect the internal organs of artificial infection rabbits. The results showed that the SYBR Green Ⅰ fluorescence quantitative PCR could quickly detect the content of the virus RNA in different organs. So this assay could be applied in clinical diagnosis of RHDV.

Key words: rabbit hemorrhagic disease virus（RHDV）, VP60, SYBR Green Ⅰ, realtime quantitative PCR