| Duck hepatitis B virus and human hepatitis B virus belong to the Hepatophilic DNA Viridae.They are similar in virus structure,viral replication process,host mechanism of infection and pathogenesis.It is helpful to study the replication cycle of hepatophilic virus and inhibit the replication of hepatophilic virus using the animal infection model of duck hepatitis B virus.The primary duck hepatocyte model infected by DHBV in vitro and the animal model infected in vivo can also be used to evaluat the role of various virus genes and screen therapeutic drugs.In this study,a novel loop-mediated isothermal amplification detection system,was established for DHBV detection.With its outstanding specificity,high sensitivity,fast and simple characteristics,a set of detection system which can not only simplify the operation process,but also meet the needs of rapid detection for grass-roots units and on-site detection is optimized and constructed.Construct and optimize the proportion of each component and raection conditions of LAMP system,an optimal LAMP reaction system was established to detect duck hepatitis B,so that duck hepatitis B virus can be detected in large quantities in grass-roots environment.After a lot of experiments,the optimum condition of LAMP detection system for duck hepatitis B virus was obtained as follows: 1.Phenol red and cresol red mixed staining solution was used as color reagent,the positive results showed bright yellow,the negative results showed purple red.Great color difference is beneficial to observation and judgment results;2.The optimum concentration of Mg ion was selected by gradient experiment.It was found that when the concentration of Mg ion was 6 mM,the amplification effect was the best;3.By adjusting the concentration of dNTPs,it was found that when the concentration of dNTPs was 1.2 mM,the effect was the best;4.In the time gradient experiment of LAMP system,the electrophoresis band with reaction time of 50 minutes was the brightest and the amplification effect was the best,so 50 minutes was the best reaction time;5.When LAMP temperature gradient experiment was carried out,it was found that the gel electrophoresis belts were brighter at 63?,64?and 65?,and the amplification effect was better.Finally,the optimum enzyme activity temperature 65? of Bst DNA polymerase was selected as the optimum temperature for the reaction.Duck serum samples collected from Nanyang duck farm,Bozhou duck farm and Qianjiang duck farm in Henan province,Anhui province and Hubei province were detected by optimized LAMP detection system,the positive samples carrying DHBV were screened out.the results showed that the natural infection rates of duck hepatitis B were 17.6%,13.6% and 16.1% in Nanyang,Bozhou,and Qianjiang,respectively,with no significant difference(P > 0.05).One DHBV positive sample from three places was selected for cloning,sequencing and sequence analysis of the whole DHBV gene,The genome length of the three DHBV isolates from Nanyang,Bozhou and Qianjiang were 3024,3027 and 3024 bp.Sequence analysis data showed that the nucleotide homology among three DHBV strains was 95.1%~97.4%,and 90.3%~96.2% with 25 selected reference strains.The analysis of genetic evolution tree and P protein amino acid key loci showed that two isolates from Nanyang,Henan and Bozhou,Anhui were Chinese-like genotypes,while Qianjiang isolates from Hubei were not Chinese-type and Western-type,and belonged to independent small branches,which might lead to viral recombination. |
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