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Application of Sanger sequencing in detection of drug-resistant mutations in patients with hepatitis B

MA Liang;YU Yang;ZHANG Jingying;CONG Xiao;LIU Qian;YANG Hui;CAO Yongtong;Department of Clinical Laboratory,China-Japan Friendship Hospital;  
Objective To analyze the application of Sanger sequencing in the detection of hepatitis B virus drug resistance and genotype,and to further explore the occurrence of six nucleotide drug resistance in hepatitis B,including tibivudine(LdT),adefovir(ADV),lamivudine(LAM),entracycline(FTC),tenofovir(TDF)and entecavir(ETV).To provide reference for clinical medication.Methods The serum samples of 215 patients with HBV-DNA copies greater than 103 in China-Japan Friendship Hospital were collected.The HBVDNA in serum was extracted and amplified by PCR.The PCR products were sequenced by Sanger sequencing.The results were analyzed and compared by STANFORD software.SPSS17.0 software was used for statistical analysis.Results A total of 40 patients(18.6%,40/215)were found to have drug resistance gene mutations,of which 21(9.77%,21/215)had corresponding reference value.Tibivudine(9.77%,21/215),entecavir(7.44%,16/215),entecavir(6.98%,15/215),tibivudine(6.05%,13/215)and adefovir(0.93%,2/215)were the most common drug-resistant mutations in the 215 patients.The incidence of genotype C was significantly higher than that of genotype B in 215 patients(P0.01).There was no significant difference between genotype C and genotype B(P0.05).Conclusion This study suggests that the mutation of HBV resistance gene can be analyzed comprehensively by Sanger sequencing,and this study also suggests the occurrence of high drug resistance of LAM drugs as the first choice for the treatment of chronic hepatitis B(CHB).
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