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Construction Of FMDV-VSV-SVDV Microarray

Posted on:2016-03-03Degree:MasterType:Thesis
Country:ChinaCandidate:Y D OuFull Text:PDF
GTID:2283330482975369Subject:Prevention of Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Foot-and-mouth disease (FMD), swine vesicular disease (SVD) and vesicular stomatitis (VS), which result in acute, violent, infectious disease of the mammal, are caused by foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV) and vesicular stomatitis virus (VSV), respectively. Since the three diseases are clinically similar to each other, it is unable to distinguish from clinical symptoms. Hence construct a high through detection method which could simultaneous detect the three disease and three serotypes of FMDV is of great significance. In this study, a detection system of 5 kinds virus oligonucleotide microarray was established which used FMDV-O, FMDV-A, FMDV-Asia 1, SVDV and VSV as the research object. The main studies were as following:1% The construction of target gene primers and probesReference in the published genome sequences of Gen Bank, the conservative region was selected to design the primers and probes which according to the design requirement of gene chip primer. Through the verification of the primers and probes, the pMD19-T cloning vector plasmids of FMDV-A, FMDV-Asia 1, FMDV-O, SVDV and VSV was been constructed and sequenced. The results showed, the consistency of clone target gene and the sequence which released NCBI is more than 95%.2% Construction and optimization of the gene chipThe matrix of genechips, position control, hybrid control and the probe of hybrid control sequence were been designed, and probes of five viruses were screened. The hydration temperature, closed condition, annealing temperature and cycle number of PCR, usage amount of primer with fluorescent groups and hybridization temperature were optimized. Experimental results showed:the best effect of gene chip was been made under these conditions:using the aldehyde substrate, hydration at 18-37℃ overnight, using sealing liquid which contains 0.25%BH4Na and 25% ethanol; choosing 58℃ of annealing temperature for PCR amplification,2μL primer with fluorescent groups,30 cycles could get the enough product, in order to improve the sensitivity in clinical detection,4-8μL primer with fluorescent groups and 40-50 could be used. Even through, above the 42℃ of hybrid temperature could obtain meaningful results, to ensure the specificity of hybridization, it could be carried out at 52℃.3、Methodology research of the 3 detect methodsTests of sensitivity, specificity, repeatability and stability were been proceeded to the microarray. Results showed:the PCR LOD (limit of detection) of FMDV-O, FMDV-A, FMDV-Asia 1, VSV, SVDV was 0.0001180u.g/mL,0.0000184u.g/mL,0.000129μg/mL, 0.0000267μg/mL,0.0000247μg/mL, respectively, the sensitivity the chip detection is at least an order of magnitude higher than that of PCR detection; the chip detection showed good specificity and accuracy in distinguish FMDV, VSV, SVDV and the three serotype of FMDV without nonspecific amplification of designed primer; the experiment showed good repeatability in different batch of aldehyde substrate which produced by two companies; the prepared chip could be saved at least four months in 4℃ after vacuum pumping.
Keywords/Search Tags:foot-and-mouth disease, swine vesicular disease, vesicular stomatitis, genechips, joint inspection, genotyping
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