cDNA文库法在HCV-1b检测芯片研制中的应用  

HCV Genotype 1b Microarray Preparation Using cDNA Fragments Library

作　　者：孙朝晖[1] 郑文岭[2] 彭翼飞[1] 张宝[1] 吕梁[1] 马晓冬[1] 马文丽[1] 

机构地区：[1]南方医科大学基因工程研究所,广州510515 [2]广州军区广州总医院肿瘤分子生物学研究所,广州510010

出　　处：《中国生物化学与分子生物学报》2005年第3期415-420,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.39880032);广州市重大科技攻关项目(No.01Z00501)~~

摘　　要：制备丙型肝炎病毒(HCV) 1b亚型诊断芯片并进行初步验证评价.采用cDNA文库法制备探针,用限制性内切酶Sau3AⅠ消化HCV 1b全长cDNA ,所得的酶切片段72℃补平加A ,AT克隆,PCR初步鉴定,并测序.将筛选出的片段打印在氨基修饰的玻片上制备成检测芯片并进行杂交验证分析.运用cDNA文库法,得到2 2个大小相对一致(2 5 0～75 0bp)的基因片段.序列分析表明,均属于HCV 1b基因,可以作为诊断芯片探针;样品标记采用限制性显示(restrictiondisplay ,RD)技术,标记后进行杂交.杂交结果显示,样品和诊断基因芯片杂交的敏感性和特异性均佳.批内和批间精密度CV值分别为5 4 %和6 8% ,表明用cDNA文库法收集片段是一种快速、简便制备芯片探针的实用方法.In order to investigate the application of cDNA fragments library for preparation of HCV-1b microarray,full-length HCV-1 sequence of subtype b was digested with restriction endonuclease Sau3A Ⅰ and the resulting fragments were cloned into the pMD18-T vectors. Positive clones were isolated and identified by sequencing. The cDNA microarray was prepared by spotting the gene fragment to the surface of amino-modified glass slides by the robotics. Restriction display PCR (RD-PCR) was used to label the samples isolated from HCV-1b or 1a patients,the different fluorescent-labeled probes were applied to microarray.Fluorescent images of the hybridized microarrays were scanned with a fluorescence laser scanner. The detection of microarray was validated by the hybridization and the sequence analysis. A total of 22 different fragments ranging from 250 to 750 bp were isolated and sequenced, which were the specific gene fragments of HCV-1b. These fragments could be further used as probes in the microarray preparations.From the results of hybridization and sequence date analysis, the specificity and sensitivity in detecting HCV-1b RNA was verified. The within and between assay coefficient of variations were 5.4% and 6.8%,respectively.It was an effective,quick and simple method for preparing microarray probes by construction of cDNA fragments library.

关 键 词：丙型肝炎病毒 限制性显示 分子克隆 基因芯片 杂交 诊断 

分 类 号：R394.2[医药卫生—医学遗传学]