Study Of The Detection Method For Rabbit Hemorrhagic Disease Utilizing TaqMan-based Fluorescence Quantitative PCR And Rabbits And Sucking Mice Infected Test

Rabbit Hemorrhagic Disease (RHD) is caused by Rabbit hemorrhagic disease virus (RHDV) which is infected adult rabbits mostly and caused high fatality and infection rate, which threaten to the blossom of the rabbit industry. Because there are no stable cells for RHDV growing, which affect the study of infection mechanism and the prevention of the disease and the emergence and epidemic of new mutant strain (RHDV2), which brings new difficulties for prevention of RHD. In this study, a real-time Taq-Man fluorescence quantitative PCR assay was established which could also identify with the RHDV variant strain. Furthermore, the assay was applied to the rabbits and sucking mice infected test and the specific method is as follows:In this study, RHDV VP60 protein partial genes which has the length of 998bp was chose to cloned as the templates and a pair of primers and a probe were designed to detect the RHDV by fluorescence quantitative PCR which was included to the templates VP60 protein gene clone and the purpose fragment length wasl73bp. The probe was signed FAM in 5'and TAMRA in 3'. Fluorescence quantitative PCR method was established and the reaction system was optimized and the standard curve was built in the end. The sensitivity test showed that the minimum that can be detected was 28copies/?L and there are no others virus were detected through this method, which means that the method had high sensitivity and specificity and it could be able to apply to detect the virus content in the various organ in samples.In this article,10 rabbits were bought in the market that were no immune RHDV before and used for the infected test.10 rabbits were dead in 3 days. All the main organs of the rabbits were collected to make and observerd pathological section and 0.2g organs were used for detecting by the q-PCR and the positive detection rate was 100%. Also, the organ virus content order was serum, erythrocyte, liver, spleen, heart, kidney, lungs and the muscle.At the same time, sucking mice were infected RHDV in the back to detect RHDV and the sucking mice organs primary cells were tocultivated and after infetcted RHDV to detect. The results indicated that the mice were dead in 24h. Then, all the sucking mice main organs were collected 0.1g to detect the RHDV by q-PCR and there were RHDV in the all organs and the higher content organs were kidney and lungs. After infected RHDV, cells were digested from the cell plate to detect the RHDV content, but there were suspected virus found in the cells except trace virus were found in kidney cells. At last, the above research laid a solid foundation and provided some references to the research of RHDV infection mechanism.