Detection Of Nucleic Acid Of Poultry Virus With Nucleic Acid Probe And Molecule Dot Blot Hybridization Technique

It is a common phenomenon that different virus in the same individual, so how to detect different virus in the same individual at the same time is very important for disease diagnosis and epidemiology research. This research attempted to use molecule dot blot hybridization technique to detect different virus in the same specimen with different nucleic acid probe at the same time, They are REV,MDV,CAV,ARV and IBV.This experiment collected 200 portion spleen samples and 200 portion feather bladder samples of the local variety chicken from one Taian market; collected 200 portion spleen samples and 100 portion chicken embryo samples from one commercial meat chicken farm in Laiyang, counted up to 700 portion samples. Detect REV,MDV,CAV,ARV and IBV with nucleic acid probe labeled in LTR sequence and pol gene of REV, nucleic acid probe labeled in pp38 gene of MDV, nucleic acid probe labeled in VP1 gene of CAV, nucleic probe labeled inδ3 gene of ARV, nucleic acid probe labeled in N gene of IBV, the result as follows:The detection result of MDV,CAV,ARV,IBV and REV in 200 portion spleen samples of the local variety chicken: MDV positive 123 portion, CAV positive 91 portion, ARV positive 21 portion, IBV positive 123 portion, REV positive 194 portion detected with nucleic acid labeled in LTR sequence, REV positive 10 portion detected with nucleic acid probe labeled in pol gene.MDV,CAV,ARV,IBV and REV co-infection situation in 200 portion spleen samples of the local variety chicken: MDV and REV co-infection 7 portion, MDV and CAV co-infection 60 portion, MDV and ARV co-infection 17 portion, MDV and IBV co-infection 10 portion, CAV and REV co-infection 9 portion, CAV and ARV co-infection 14 portion, CAV and IBV co-infection 13 portion, MDV,CAV and REV co-infection 6 portion, MDV,CAV and ARV co-infection 13 portion, MDV,CAV and IBV co-infection 7 portion ( The data of REV infection comes from the detection result with the nucleic acid probe labeled in pol gene ). MDV,CAV,ARV,IBV and REV infection situation in 200 portion feather bladder samples: CAV,ARV and IBV all negative, MDV positive 33 portion, REV negative detected with nucleic acid probe labeled in LTR sequence and pol gene.The detection result of MDV,CAV,ARV,IBV and REV in 200 portion spleen samples of the commercial meat chicken: MDV positive 177 portion, CAV positive 143 portion, ARV and IBV negative, REV positive 103 portion detected with nucleic acid probe labeled in LTR sequence, REV negative detected with nucleic acid probe labeled in pol gene.MDV,CAV,ARV,IBV and REV co-infection situation in 200 portion spleen samples of the commercial meat chicken: MDV and CAV co-infection 137 portion (The data of REV infection comes from the detection result with the nucleic acid probe labeled in pol gene ).The detection result of MDV,CAV,ARV,IBV and REV in 100 portion Laiyang chicken embryo samples: MDV,ARV and IBV all negative, CAV positive 100 portion, REV negative detected with nucleic acid probe labeled in LTR sequence and pol gene.When detected REV from 200 portion spleen samples of the local variety chicken and 200 portion spleen samples of the commercial meat chicken with nucleic acid probe labeled in LTR sequence and pol gene, the difference between the two nucleic acid probe is significantly. In 200 portion spleen samples of the local variety chicken, REV positive 194 portion detected with nucleic acid probe labeled in LTR sequence, REV positive 10 portion detected with nucleic acid probe labeled in pol gene, and 194 positive samples include this 10 positive samples; in 200 portion spleen samples of the commercial meat chicken, REV positive 103 portion detected with nucleic acid probe labeled in LTR sequence, and REV negative detected with nucleic acid probe labeled in pol gene. The result shows: most of the result of REV detected with nucleic acid probe labeled in LTR sequence is non-specific, the false positive of REV is made possiblely by LTR sequence integrates into genome of other viruses or host animals. So can't use nucleic acid probe labeled in LTR sequence when detected REV with dot blot hybridization, should choose other gene such as pol gene, in this way can improve specificity when detect REV.Extract DNA and RNA from bone marrow,thymus and bursal of disease chicken from Laiyang, detect REV,MDV,CAV,ARV and IBV with dot blot hybridization. dot blot hybridization shows CAV positive, REV,MDV,ARV and IBV all negative. Design a pair of primer, the VP1 gene fragment was identified,sequenced and analyzed. The result of sequence analysis showed that the VP1 gene is 1350bp. Sequence comparison of the VP1 gene from different virus strains showed that VP1 of CAV in specimen is highest with America strain.