寡核苷酸芯片技术检测乙肝病毒拉米夫定耐药株的应用研究

目的建立乙肝病毒拉米夫定耐药检测寡核苷酸芯片技术,并对乙肝病毒拉米夫定耐药检测寡核苷酸芯片的临床应用进行了评价。方法通过对388份服用拉米夫定、559份未服用拉米夫定的HBV DNA阳性血清以及359份HBV DNA阴性血清进行耐药突变检测,同时对照进行荧光定量PCR检测和DNA序列测定,对芯片法检测的准确度、实用性进行考评。结果对388份乙型肝炎病毒DNA多聚酶突变标本均能检测到突变;528、552、555位密码子突变检测芯片法与测序法的符合率分别为96.6%,98.5%,100%。559例荧光PCR检测HBV DNA阳性的样品,除3份弱阳性标本外,均为阳性,且能进行乙型肝炎病毒DNA多聚酶拉米夫定耐药相关位点的检测,两者的符合率为99.7%。359份HBsAg阴性标本经芯片法检测,结果为HBV DNA全部阴性。结论乙肝病毒拉米夫定耐药检测寡核苷酸芯片可用于临床检测病人血清中的野生型和拉米夫定耐药突变型病毒株,指导临床用药。

Application Study of Oligochips Detecting HBV and Lamivudine Drug Resistance

Objective To establish a oligochip method for detection of HBV Lamivudine drug resistance and evaluate the clinical role of the chip system.Methods 388 HBV DNA positive sera from patients receiving lamivudine treatment and 559 chronic hepatitis B patients not receiving lamivudine treatment,and 359 sera from HBV DNA negative controls were assayed for HBV mutations utilizing oligonucleotide microarray.Meanwhile,these results were contrasted with Quantitative PCR and DNA sequencing method.Results The results of clinical evaluation shows that for the codons 528,552 and 555,the agreements between the microarray and sequencing data are 96.6%,98.5% and 100%,respectively.In the 559 samples,which were detected positive for HBV DNA by quantitative PCR,all but three weak positive samples were positive by the microarray,demonstrating an agreement of 99.7%.All the 359 HBsAg negative samples were shown to be negative for HBV DNA by the microarray method.Conclusions The HBV-Lamivudine oligochip is eligible to detecting wild type HBV and HBV lamivudine-mutants in patient's sera.It has great potential application of administration for lamivudine treatment in HBV patients.