Development of a real-time RT-PCR for Rabbit Hemorrhagic Disease Virus detection according to VP10 gene

Rabbit hemorrhagic disease virus (RHDV) is an acute, severe, highly infectious and highly fatal disease in rabbits. To establish an efficient and rapid method for detecting Rabbit Hemorrhagic Disease (RHDV) by SYBR Green real-time PCR. Specific primers were designed according to the conservative sequence of VP10 gene of RHDV. A quantitative PCR method for detection of RHDV was established after a serial of tests such as Optimum of reaction conditions, sensitivity and specificity test, repeatability test and clinical samples detection. The results showed that the standard concentration had a good linear relationship in the range of 1.20x10(exp 7) - 1.20x10(exp 2) copies/muL, and the correlation coefficient was R(exp 2) = 0.999. The lowest positive plasmid could be detected in the range of 1.20 copies/muL. The method had no crossreaction with Rabbits Pasteurella, Rabbits Escherichia coli, Rabbits Salmonella and RHDV2 gene fragments that constructed by overlapping PCR. Number (CV) and coefficient of variation between batches were less than 3%. The results of clinical samples were higher than those of ordinary RT-PCR. These results showed that the fluorescence quantitative PCR method established in this study has the advantages of high sensitivity, specificity, stability, high accuracy and fast detection. And this method can be used for early diagnosis of rabbit hemorrhagic disease, and rapid and quantitative analysis of RHDV samples.