摘要
目的:制备一款肝炎病毒检测芯片,能同时实现对乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、HBV YMDD变异株及HCV基因型的检测,并与其他检测方法进行比较,以探讨基因芯片技术临床应用的可行性. 方法:根据HBV、HCV的序列设计出探针,采用点样法制备芯片.收集HBV DNA阳性和HCV RNA阳性血清各20 份,YMDD变异株阳性血清20份,HBV DNA阴性和HCV RNA阴性血清各10份,用基因诊断芯片检测,并与荧光定量法、错配PCR及测序法比较. 结果:HBV DNA阳性标本和HCV RNA阳性标本用芯片检测均为阳性;芯片法检测HBV YMDD变异株和错配PCR法的符合率为75%,和测序法的符合率为95%;芯片法检测HCV基因型和测序法的符合率为75%. 结论:基因诊断芯片的敏感性和特异性较高,且能检测出不同病毒株的共生状态,但在检测HCV基因型方面存在一定的假阴性和假阳性.
AIM: To develop a DNA microarray to detect hepatitis virus B (HBV) DNA, hepatitis virus C (HCV) RNA, HBV YMDD mutant and HCV genotype simultaneously. At the same time, the chip was compared with other techniques to evaluate its prospect in clinical application. METHODS: A set of probes was designed to detect HBV DNA, HCV RNA, HBV YMDD mutant and HCV genotype. The probes were synthesized by DNA synthesizer. The microarray was prepared by spotting the probes onto the specially treated glass sliders. Serum samples were collected from inpatients and outpatients at the Department of Infectious Diseases, the Third Affiliated Hospital of Zhongshan University. Among the samples, 20 were comfirmed HBV DNA positive by fluorescent quantitation PCR, 20 were HCV RNA positive, 20 were comfirmed YMDD mutant by mismatched PCR, 10 were HBV DNA and HCV RNA negative. HBV DNA and HCV RNA were extracted from the serum, then amplified by asymmetric PCR or RT-PCR in the presence of sense fluorescein labeled primers. The products of HBV YMDD and HCV NS-5 were purified and sequenced. Following the hybridization of amplified products on the microarrays, detection was carried out by the fluorescence scanner. The detection results were obtained by analyzing the intensity and ratio of the fluorescence signals using image analysis software. RESULTS: For the HBV DNA positive samples and HCV RNA positive samples, an intensive signal was observed at the point of corresponding probes on the microarrays. In detection of YMDD mutant, the coincident rate of the microarray and the mismatched PCR was 75%, the coincident rate of microarray and sequencing was 95%. In detection of HCV genotype, the coincident rate of microarray and sequencing was 75%. CONCLUSION: The technology of microarray appears to be versatile, with a great sensitivity and specifity in detection of HBV and HCV. Furthermore, it can find co-infection of different virus strains. But it has some false negative rate and false positive rate in HCV genotyping.
出处
《世界华人消化杂志》
CAS
2004年第4期866-870,共5页
World Chinese Journal of Digestology
基金
广东省科技重点项目资助
No.2km05302S~~
