Evaluation of PCR conditions for the analyses of specific regions of Hepatitis B Virus
LI Min;JIANG Feifei;ZHAO Yang;LIN Hongkeng;QIAN Rong;WANG Lunshan;LV Rong;Anhui Blood Center;Fujian Blood Center;Jiangxi Blood Center;PLA 105th Hospital;
Objective To evaluate the PCR conditions and to analyze the sequences in specific regions of Hepatitis B Virus( HBV). Methods Samples from six hepatitis B patients were carried out by PCR or Nest PCR methods. The volume of reagents in PCR amplification,such as the concentration of magnesium( Mg2 +),primers,Taq DNA polymerase and dimethyl sulfoxide( DMSO),were adjusted to achieve the specific amplicons of HBV. The specific fragments were directly sequenced and analyzed by CHROMAS and MEGA applications. Results In the 50 μL PCR reaction mixture,2. 5 m M Mg2 +,200 n M specific primers and 1. 5 U Taq DNA polymerase at 56 ℃ annealing temperature could achieve the best results in HBV amplification. DMSO should be properly used in large fragment and DNA polymerase can be appropriately reduced when getting shorter fragments. Among the 6 patients,1 sample was identified as genotype B and 5 as genotype C.Twenty-one nucleotide variations and 9 amino acid mutations were observed in HBV S gene of these samples. Conclusion PCR amplification of specific HBV gene was established and the sequences of S gene were analyzed by bioinformatic applications. The concentrations of reagents in PCR mixtures are very important in obtaining specific results.
【CateGory Index】： R512.62;R440