Establishment And Application Of Diagnosis Methods Of PRV Wild Strain And Mutant Strain

According to the whole genome sequence of pseudorabies virus (PRV) inGenBank, nine pairs of primers were designed to amplify the target gene of gI, gE, gD,gG,28K genes of PRV SA virulent strain. The gene inserted into T vector, positiveclones was identified by three methods of PCR, digested with single and doublerestriction enzyme respectively. The sequence Blastn analyzed and submitted to theDDBJ/EMBL/GenBank database. Respectively AB440240(gG), AB440243(gE),AB440295(gI) and AB44024(TK).For four specific sites of gI, gE,28K, TK gene, five primers designed, SAvirulent strain, gI-/gE-/PRV SA mutant strain and Bartha K61strain was amplified,target band was2061bp,1323bp,801bp and963bp. Minimum detectableconcentration genome of Bartha K61vaccine strain was136pg/μL. Establishment testmethod of two vaccines and wild virus strain by PCR. Obtained mutant sequence bysequencing gI-/gE-/PRV SA strain, GenBank accession number GU262988.1.Clerified the exact mutant position of gI-/gE-/PRV SA strain (gI and gE deletionpartial sequences totaling738bp) and PRV Bartha K61vaccine strain (part gI gene,all of the gE and US9gene and part of the US2gene totaling3489bp).Established a SYBR Green I real-time quantitative PCR to detect pseudorabiesvirus gE gene deletion strains. PCR was more sensitive1000times than the traditional,the former to1.75copies/μL, the later to1.75×103copies/μL. This method candetect PRV, no cross-reactive associated with PCV2, PPV, CSFV, PRRSV virus. InChina’s vaccine market the six kinds of pseudorabies vaccine were tested, andfounded which complied viral genetic background. two positive gE gene PRV strainwere found in Shandong area from positive samples of PRV separating material.Some gE mutation of antigenic sites were founded after sequencing.Established a gE-ELISA method for detection,240PRV sera samples of differentfarms were detected from from Shandong, PRV gE antibody positive rate of27%,IDEXX HerdChek ELISA Kit positive rate of25.8%, positive compliance rate 95.19%.Established a PRV gD-ELISA detection method to determine the optimalreaction conditions and criteria, detected from Shandong, Henan, Hebei and otherplaces306serum samples,258were detected positive, negative58copies, serumantibody positive rate84.3%.