Chen Li-Yan, Huang Jian, Zhang Xin-Ping, Qiao Ping, Zhang Wen, Yang Ning-Min, Liu Hong-Jing, Geng Yong-Yao, Qiu Jian-Ming, Wang Sheng-Qi
Beijing Institute of Radiation Medicine, Beijing 100850, China.
Pharmacogenomics. 2005 Oct;6(7):721-30. doi: 10.2217/14622416.6.7.721.
Long-term lamivudine administration in hepatitis B virus (HBV)-infected patients induces the emergence of HBV mutants with lamivudine resistance. The aim of the present study was to evaluate the clinical application of an oligonucleotide microarray in detecting HBV mutants associated with lamivudine resistance.
947 HBV DNA-positive sera from: 388 patients receiving lamivudine treatment, 559 chronic hepatitis B patients not receiving lamivudine treatment, and 359 from HBV DNA-negative controls, were assayed for HBV mutations using the oligonucleotide microarray. Furthermore, follow-up studies were performed using 255 clinical samples from 51 patients treated with lamivudine at various periods. The results were compared with sequencing and real-time polymerase chain reaction (PCR).
The HBV DNA polymerase Tyr-Met-Asp-Asp motif (YMDD) mutation was detected in all 388 samples containing lamivudine-resistant mutations identified by microarray. For the codons rt180, rt204 and rt207, the agreements between the microarray and sequencing data are 96.6, 98.5 and 100%, respectively. Two previously unreported mutants were also found in those samples. In the 947 samples collected from different patients, 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%. In all the positive samples, mutations could be detected in the relevant loci of HBV DNA polymerase with lamivudine resistance. All of the 359 HBsAg-negative samples were shown to be negative for HBV DNA using the microarray method. Follow-up detection of the clinical samples from 51 patients treated with lamivudine demonstrated that the microarray method was able to detect mutations in mixed viruses that were infecting prior to sequencing.
The oligonucleotide microarray can be conveniently utilized to detect mutant HBV in clinical serum samples.
对感染乙型肝炎病毒（HBV）的患者长期给予拉米夫定治疗会诱导产生对拉米夫定耐药的HBV突变体。本研究的目的是评估寡核苷酸微阵列在检测与拉米夫定耐药相关的HBV突变体中的临床应用。
使用寡核苷酸微阵列对947份HBV DNA阳性血清进行HBV突变检测，这些血清来自388例接受拉米夫定治疗的患者、559例未接受拉米夫定治疗的慢性乙型肝炎患者以及359例HBV DNA阴性对照者。此外，对51例接受拉米夫定治疗不同时期的患者的255份临床样本进行了随访研究。将结果与测序和实时聚合酶链反应（PCR）进行比较。
在通过微阵列鉴定出的所有388份含有拉米夫定耐药突变的样本中均检测到HBV DNA聚合酶酪氨酸-甲硫氨酸-天冬氨酸-天冬氨酸基序（YMDD）突变。对于密码子rt180、rt204和rt207，微阵列与测序数据之间的一致性分别为96.6%、98.5%和100%。在这些样本中还发现了两个以前未报道的突变体。在通过定量PCR检测出HBV DNA阳性的947份来自不同患者的样本中，除三份弱阳性样本外，其余样本通过微阵列检测均为阳性，一致性为99.7%。在所有阳性样本中，均可在具有拉米夫定耐药性的HBV DNA聚合酶相关位点检测到突变。使用微阵列方法显示，所有359份HBsAg阴性样本的HBV DNA均为阴性。对51例接受拉米夫定治疗患者的临床样本进行随访检测表明，微阵列方法能够在测序之前检测出感染的混合病毒中的突变。
寡核苷酸微阵列可方便地用于检测临床血清样本中的突变型HBV。
