Abstract-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℃ 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.
Abstract-To develop a rapid method for the detection RHDV2, 4 specific primers and 12 overlapping PCR primers were designed to amplify the conserved RHDV2 specific DNA fragment according to the genome sequences of RHDV and RHDV2 published in GenBank. Based on the synthesis of a conserved part of the RHDV2 sequence using overlap extension PCR, a SYBR Green I RT-qPCR assay was first preliminary developed and evaluated in China after a series of tests, including, reaction conditions optimization, establishment of standard curve, the sensitivity and specificity tests, intra-assay , and the application tests of 30 samples. The results showed that a 435bp specific DNA fragment of the RHDV2 capsid protein(VP60)gene was synthesized in vitro and the recombinant plasmid pMD-19T-RHDV2 was constructed, and the RT-qPCR method for RHDV2 rapid detection had good specificity, sensitivity and repeatability, which performance was linear ranging from 128 copies/μL to 1.28×108copies/μL for the cloned viral genomic fragments of RHDV2.The sensitivity of the RT-qPCR could reach about 128 copies/μL of target fragments, and there was no amplification for RHDV, pGM-T-EBHSV, Pasteurella multocida, E.coli and Salmonella from rabbits detection by this method. And the application tests results showed that there were not RHDV2 in the experimentally infected samples and clinical samples.
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