【摘要】 目的评价焦磷酸测序法在慢性乙型肝炎(CHB)患者阿德福韦酯(ADV)耐药检测中的应用。方法选取ADV治疗失败的CHB患者,采用PCR产物焦磷酸测序法检测ADV耐药相关的rtA181V/T和rtN236T变异,并与PCR产物双脱氧测序法进行比较。计算两种方法的耐药检出率及采用焦磷酸测序法检出的变异株在病毒群中所占的比例。结果共入选患者210例,经双脱氧测序法与焦磷酸测序法检测共确认耐药变异患者67例,耐药变异位点92个,其中焦磷酸测序法检出耐药患者64例(95.52%)、变异位点86个(93.48%),双脱氧测序法检出耐药患者52例(77.61%)、变异位点71个(77.17%),两种方法结果符合率为83.8%。焦磷酸测序法检出44例发生单位点变异的患者,其中变异株比例<50%的患者26例,最低耐药株比例为6.5%。rtA181T、rtA181V、rtN236T变异的平均变异株比例分别为34.12%、61.23%、59.69%,其中rtA181V与rtN236T的变异株比例无明显差异(P=0.909),但均高于rtA181T的变异株比例(P<0.01,P<0.05)。结论焦磷酸测序法的ADV耐药检出率优于双脱氧测序法,前者可进一步计算患者体内变异株的比例;在大部分患者中检出耐药时变异株不一定是优势株。更多还原

【Abstract】 Objective To evaluate the application of pyrosequencing in the detection of HBV genotypic resistance to adefovir dipivoxil(AD)in chronic hepatitis B(CHB)patients.Methods Serum of CHB patients who failed in AD mono-therapy(including patients underwent the viral breakthrough and patients with HBV still higher than the low-detection limit after at least 48-week therapy)were collected.AD associated mutations including rtA181V/T and rtN236T were detected by pyrosequencing,which was compared with the results of dideoxy sequencing.The detection rate of drug resistance and the ratio of mutants detected by pyrosequencing in virus group were calculated with these two methods.Results Tow hundred and ten CHB patients were internalized.Totally 67 patients with AD resistance and 92 mutation sites were detected,including 64 patients(95.5%)with AD resistance positive and 86(93.5%)mutation sites detected by pyrosequencing,and 52 patients(77.6%)with AD resistance positive and 71(77.2%)mutation sites detected by dideoxy sequencing.The coincidence of the two approaches was 83.8%.There were 44 patients with single mutants,26 of them had a mutant rate less than 50%,and the lowest rate was 6.5%.The mean mutant rate of rtA181T,rtA181V and rtN236T was 34.12%,61.23% and 59.69%,respectively.There was no difference between rtA181V and rtN236T in mean mutants rates(P=0.009),while both of the two pattern of mutations were higher than that of rtA181T(P<0.01或P<0.05).Conclusions The detection rate of AD is higher detected by pyrosequencing than by dideoxy sequencing,and the former can be also used in the calculation of mutant ratio.The variant when drug resistance is detected in most patients may not be the dominant species.更多还原