Preliminary Study on The RT-LAMP Assay for Rabbit Hemorrhagic Disease Virus Type 2 Detection

摘要

To develop a rapid molecular biological method for the detection of rabbit hemorrhagic disease virus type 2 (RHDV2), a set of 4 primers were designed according to the conserved sequence fragment of the capsid protein (VP60) gene of RHDV2 published in GenBank, and the reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was established through identification of target gene fragments, optimization of reaction conditions, sensitivity and specificity tests, and the application tests of 30 samples. The results showed the RT-LAMP method for the RHDV2 detection had a ladder-like pattern of amplification bands from about 119 bp incubation at 63.5°C for 90 min by using agarose gel electrophoresis, the sensitivity of the RT-LAMP could reach about 180 copies/μL of target fragments, there was no amplification for RHDV, pGM-T-EBHSV, Pasteurella multocida, E.coli and Salmonella from rabbits detection by this approach. The application tests results showed that there were not RHDV2 in the experimentally infected samples and clinical samples. The RT-LAMP assay established here had good specificity and sensitivity, which is suitable for RHDV2 rapid detection.