Study And Tentative Application Of The Detection Method For RHDV Utilizing Real-time PCR

Rabbit hemorrhagic disease (RHD)is an acute and contagious rabbit infectious disease, due to high morbidity and mortality,which was caused by Rabbit hemorrhagic disease virus (RHDV). There is not an perfect detect method so far.The routine detection of RHDV is HA, HI, IHA, AGP, ELISA, RT-PCR et al. These methods have some disadvantages, for example, low speciality, time-consuming, low sensitivity, fake positiveity et al.Therefore, it is important for us to test a rapid and exact detection method.The real-time PCR is a new detection of nucleic acid, which is used to bacterial, virus, and genictic disease in the clinical detection. Based on the fluorescence quantitative PCR technology, referenced rabbit hemorrhage disease virus's(RHDV)conservative gene VP60, designed primers and Taqman MGB probe.The method has used in detecting the rabbit hemorrhage disease virus by real-time RT-PCR.Three experiments were carried out to obtain the study.Experiment 1,the clone of part VP60 of RHDV protein. RNA of the RHDV was extracted and reverse transcripted.The production of reverse transcription was amplified by PCR using the designed primers.The electrophore of the PCR production in 1% agarose indicated that the amplifictor is about 1201bp.The amplifictor is purified and joined into the pUCm-T Vector,then transformated into the E.coli DH5α.After screened by...