Establishment On Detecting To Four Egg Transmission Of Virus Diseases By Multi-PCR

In recent years, with the rapid development of large-scale farming, egg transmission of virus disease such as Reticuloendotheliosis virus (REV),chicken infectious anemia virus (CAV), avian leukemia viruses (ALV), avian Reovirus (ARV), avian encephalomyelitis virus (AEV) was prevalent in chickens in China and was co-infected, it caused huge economic losses to the poultry industry. These egg transmission of viruses can not noly cause parental disease directly or chronic persistent infection, or chronic cancer, or immune suppression and other hazards, more importantly, virus can be transmissed directly by eggs form a new round of infection. The disastrous consequences would be incredible if the disease was transmissed by SPF chicken and was contaminated by chicken embryo vaccine. Therefore, this study was to establish a kind of fast, accurate and sensitive detection method for chicken REV, CAV, ALV and ARV four common egg transmission viruses.In the first place, experimented in proliferator of REV,CAV,ALV and ARV, REV and ALV were proliferated by chick embryo fibroblast(CEF),ARV and CAV were proliferated by SPF chick embryo. The correlative genes of the four viruses were took homology analysis respectively, screened out PCR amplification primer sequence involved the LTR gene sequence of REV HA9901 strian,the VP3 gene sequence of CAV SD24 strian,the GP85 gene sequence of ALV HPRS103 strian and the S4 gene sequence of ARVs1113 strian,single PCR detection method of REV,CAV,ALV and ARV was established.The created method was optimized such as annealing temperature,Mg2~+ concentration,primer concentration and Taq concentration;the result shows that the primer amplification fragment size were 409bp,580bp,1260bp and 207bp, annealing temperature,Mg2~+ concentration,primer concentration and Taq concentration were 53℃,1.5μL,1.5μL,0.5μL. After sequence analysis,sensitivity tests,specific tests and repeatable test, The results show that the established single PCR detection method could be used for detecting the clinical disease material,cytotoxic and embryo toxicity.In the second place,four PCR of REV-CAV-ALV-ARV was established based on the established single PCR. The created method was optimized such as annealing temperature,Mg2~+ concentration,primer concentration and Taq concentration. result shows that annealing temperature,Mg2~+ concentration,primer concentration and Taq concentration were 53℃,1.5μL,1.5μL,0.5μL. 30 suspicious disease material from anhui province were amplified by single PCR, reviewed by four PCR, the result shows that the positive coincidence rate was 93% and the four PCR could be used detection.Finally, 80 clinical material and 100 eggs from five farms were detected by the method, the result shows that the four egg transmission virus diseases were prevalent in chickens and mixed infection was serious;114 poultry live vaccine of embryos produced were detected by the method, the result shows that there are three batches positive for CAV and two batches positive for ALV; 120 SPF chicken embryo were tested only two were detected positive for ALV. Preliminary applications show that the establishment of this study for four PCR about REV,CAV,ALV and ARV four common virus egg transmission, the method can be used for clinical diagnosis, epidemiological investigation, poultry vaccine testing of embryo, with important applications value, to achieve the desired goal.