摘要
[目的]利用膜芯片技术建立乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)联合诊断和基因分型新方法。[方法]根据HBV的前C区、X区及HCV的5'端非编码区(5'UTR)基因序列分别设计引物与分型探针。用生物素标记的引物进行HBV-DNA和HCV-cDNA扩增,将活性氨基标记的分型探针依次固定在尼龙膜上,制成HBV和HCV联合诊断和基因分型膜芯片条。扩增产物与膜芯片条杂交后,经过氧化物酶与底物显色,判断有无HBV和HCV感染并同时进行基因分型。通过经荧光定量PCR和基因测序确定的HBV和HCV单项阳性血清各30份以及HBV和HCV混合血清5份验证该方法的有效性。[结果]膜芯片对HBV和HCV单独感染和混合感染的诊断与荧光定量PCR结果相同,基因分型结果与测序分型结果一致。[结论]利用膜芯片技术可实现HBV和HCV联合诊断和基因分型,并具有准确有效和简便经济等特点,适合用于临床诊断和流行病学调查。
[Objective] To establish genotyping and diagnosis methods for hepatitis B virus （HBV） and hepatitis C virus （HCV） using gene chip technique . [Methods] Primers and genotyping probes were designed in region X and region pro-C of HBV and 5＇-untranslated region of HCV. Primers were biotin-labeled to amplify HBV DNA and HCV cDNA. Genotyping probes were immobilized on nylon membrane to make genotyping gene chip. The PCR products were hybridized with the genotyping gene chip. The color was developed adding POD and TMB. A judgment could be done certainly according to position of color reaction. The reliability of this new method was verified by FQ-PCR and sequencing. HBV and HCV infection could be identified and genotyped with the method we established simutaneously. 30 HBV DNA-positive, 30 HCV RNA-positive sera and 5 HBV/HCV coinfection sera were genotyped. [Results] The results of genotyping gene chip were coincided with FQ-PCR and sequence analysis. [Conclusion] HBV and HCV co-diagnosis and genotyping can be realized by gene chip technique which has proved to be sensitive, specific, precise and economic, and it is suitable in clinical practice and epidemic study.
出处
《现代预防医学》
CAS
北大核心
2007年第5期817-819,共3页
Modern Preventive Medicine
基金
佛山市科委重点科技公关项目(0008010A)
