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

Abstract

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.

Preliminary Study of LAMP Method for the Detection of Equine Infectious Anemia Virus

“…To identify the conserved target gag gene fragment of EIAV, the constructed recombinant plasmids pMD-19T-gag was identified by were subsequently identified by PCR (using F/R as primers) and by sequencing analysis as described in [17] .The PCR reaction volume (50 μL) containing 25μL of 2×Taq PCR MasterMix, 1μL (10μmol/L) of each of the primers (F/R), 1μL of recombinant plasmids and 22μL ddH 2 O. The PCR procedure program were: denaturing at 95℃ for 5 min, followed by 35 cycles at 94℃ for 40 s, 52℃ for 30 s, and 72℃ for 30 s, then terminated by an elongation at 72℃ for 10 min.…”

“…The number of target gene fragment molecule copies in each reaction was calculated according to, then the sensitivity of RT-LAMP was accessed [17] .…”

“…The amplification of gene and detection for products complete by one step [8,9] , with traits of high specificity, promote and sensitive, high efficient, and ease of operation, etc [10][11][12][13][14] . And then it received the worldwide concern [15,16] . As a novel nucleic acid amplification method, there is no reports on the LAMP methods for EIAV.…”