The Preparation Of The DNA Probe Of Chicken Inclusion Body Hepatitis Virus And Its Application In Dot Blot Hybridization

Chicken inclusion body hepatitis (IBH) is one kind of acute infectious diseases caused by the avian adenovirus. There are 12 serotypes related to the disease of avian adenovirus. The infected avian always died suddenly, autopsy shows severe anemia, jaundice, hemorrhage, necrosis and enlargement of liver, it can some times cause inclusion bodies within the liver cytoblast. The disease, also known as anemia syndrome.The target tissue of avian adenovirus is mainly liver and it could form inclusion bodies within the liver cytoblast. As it can lead to the decrease in production of poultry and Layer chicken, cause immunologic deficiency and thus contribute to the immune Failure as well as Secondary infection. As the damage caused by avian inclusion body hepatitis more and more serious day by day, the disease calls much attention from all aspect concerned. However, there is still not a way to detect the pathogen briefly, quickly and economically. Dot blot hybridization becomes one kind of ideal method because of its simple operation and cost control.Design a pair of primer according to the present reference and at the same time design another pair of primer to set as contrast. The second pair of primer is designed on the basis of the conserve sequence of Hexon gene from NCBI GenBank. Get the target production from the amplification of the liver tissue by PCR. Then, through the retrieve of the target production from the gelatin, purification of the nucleic acid, the PCR production can be cloned into the T-easy vector and thus transfer into DH5α. From the operation above, we obtain the recombinant bacteria, select twelve clone for sequencing. The result of the sequence comparison shows that there are as many as ten strains of the clone are in concord with the sequence of the serotype 8 of avian adenovirus. The homology is varying from 98%to 98.8%.Extract and restriction enzyme of the recombinant plasmid to make sure the target fragment is right. Amplification of the objective nucleic acid by PCR and then labeled it by digoxin kit. The labeled probe shows favorable sensitivity and specificity. The sensitivity assay indicates that the minimum content for detection is lpg and the specificity assay shows no cross reaction, the probe is reliable for laboratory detection. Optimizations of the system of the dot blot hybridization and detect some lesion liver tissue from some region of Shandong province. The result manifest we should pay much more attention to the avian inclusion body hepatitis.Treat with the lesion liver tissue and inoculate to the CEK cells, transfer of culture for several generation and extract the cell DNA. The nucleic acid then hybrid with the digoxin labeled probe. The detection of the pathogen illustrates the reliability of the probe further more.According to the research, we can see the widespread of the pathogen of IBH in the breed industry. The popular serotype in Shandong province is mainly serotype 8. The system of the dot blot hybridization by the digoxin labeled probe is showing well availability. It is likely to be used in the breed industry and laboratory diagnosis.