A Nover Method For Efficient Amplification Of Whole HBV Genomes And Contribution Of The Variation Of HBV To The Severe Hepatitis B

Objective: The severe hepatitis B has a high incidence rate and complex mechanism.The treatment is very difficult and has a bad results.It is a key problem of medical study.Our purpose is to establish a method which allows sensitive amplification full-length HBV genomes with less copy and to research into the function of the variation of hepatitis B virus to the severe hepatitis B have been questions for modern medical research.Methods: (l)Treatment of sampals:First,7 different methods has been used such as NaoH denaturation,sodium octanoate and phenol/chloroform ect.The result have been compared based on the obtaining rate,keeping of integral and sensitivity of amplification,ect.The treatment of samples with20% PEG8000 has shown that the method is sensitive and reproducible. (2)Primer design:For this problem,the primers for PCR which are located at or near the nick region have been chosen as these primer binding sites are highly conserved in all HBV genotypes.(3)The PCR products have been cloned into vector pUCm-T and have been analysed by PCR and sequence.Result:(1) The treatment of samples with 20% PEG8000 for the detection of HBV DNA is more sensitivity than the other method.(2) We canreproducibly obtain an products of expected size(approximately 3.12kb) with the primer pair HBVL1/ HBVL2.(3) Our best results are obtained with an elongation time of 6 min for first 25 cycles and 12 min for the last 10 cycles. (4)Two of three cases display mutations concerning the nucleotides 1762-A-Tand 1764-G-A.Conclusion: The treatment of samples with 20% PEG8000 could efficiently reduce the bilirubin and has no harm for template.With the primer pair HBVLi/HBVLa we can amplify nearly full-lengh products from a small genome copies of HBV.Double mutations in the CP at nt 1762 and nt 1764 have been found and should be further researched.