| Pseudorabies?PR?is a kind of acute and contagious disease,caused by Pseudorabies virus?PRV?.The virus can infect a variety of animals and has a high mortality rate,especially the death rate for low-age animals is about 100%.The pocine are major host and spread groups.The disease has a serious impact on the pig industry and it can spread and be infected by respiratory tract and cntacting excrement.In order to prevent the infection of this disease,the main method is to inject gE gene deletion vaccine of the PRV.However,in recent years,the effects of current vaccine has been greatly reduced,and the incidence of pseudorabies has been on the rise.Therefore,the establishment of a PRV detection method,which is capable of rapidly identifying wild-type strains and vaccine strains,is of great significance for the prevention and control of pseudorabies disease.In this experiment,I designed primers for the major virulence gene gE of pseudorabies virus wild type and pseudorabies virus conserved gene gB,optimized the PCR reaction system to establish a clinically applicable duplex PCR detection method for PRV,and applied the duplex PCR method to detect clinical samples for understanding PRV infection in Changchun.The results are as follows:1 The establishment of duplex PCR for PRVIn this experiment,gE primers and gB primers were added to the same reaction system based on the single-PCR reaction to perform synchronous amplification of the gE gene and gB gene fragment.In the duplex PCR reaction,the two pairs of primers were mixed and subjected to fragments amplification of gE gene and gB gene.The primers that did not interfere with the amplification were selected.The enzyme,dNTP and magnesium ion's concentration,and annealing temperature were optimized separately.Finally,the determination of the 25 ul reaction system---the enzyme?0.05units/ul?,dNTP?0.15mM each?,magnesium ion?1.5mM?,annealing temperature?60??---of duplex PCR reaction conditions is the best suitable.The specificity,sensitivity,reproducibility and conformity to the experiment demonstrated that the specificity of the duplex PCR is high and the stability is good.The method can amplify DNA with a concentration of 2.003×10-5ng/ul or more.The positive detection rate is 100%.2 The application of duplex PCR for PRVIn 2017,167 samples of suspected pseudorabies were collected from pig farms in Changchun and surrounding areas,of which 54 were from February to April,26 from May to July,70 from August to October,and 17 from November to December.The duplex PCR method was used for detection.The positive result of wild-type virus was 58 and the positive rate was34.73%.The positive rate was 35.19%and 35.71%in spring and autumn,respectively.The positive rate was the lowest in winter?29.41%?,compared with 34.62%in summer.The incidence of pseudorabies in the region of Changchun is relatively serious and can occur in all seasons,and the PRV wild-type virus infectionin Changchun region is on the rise.This study provided a reference for the prevention and control of pseudorabies infection in Changchun. |
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