丙型肝炎病毒1b亚型诊断芯片的制备与实验室研究

The study in preparation of HCV-1b cDNA microarrays probes

目的建立一种简单有效的制备、筛选丙型肝炎病毒(HCV)1b亚型cDNA基因芯片探针的技术.方法应用cDNA文库法制备芯片探针,限制性内切酶Sau3AⅠ消化HCV-1b全长cDNA,所得的酶切片段在72℃补平加单个碱基A,然后与pMD18-T载体连接,AT克隆,用载体引物进行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cDNA芯片并进行杂交验证分析.样品标记采用限制性显示PCR(Restriction Display PCR RD-PCR)技术.结果应用cDNA文库法,共得到22大小相对一致(250～750bp)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;芯片杂交结果显示,样品和诊断基因芯片杂交的敏感性和特异性均佳.结论用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;制备的诊断芯片可以用于检测HCV-1b RNA,具有敏感、检测结果较为可靠的优点.

Objective To develop a simplified and efficient method for HCV 1b cDNA microarray probes preparation and collection Methods The full length HCV cDNAs subtypes of 1b were 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 amido modified glass slides by the robotics Restriction Display PCR (RD PCR) was used to label the samples The detection of microarray was validated by the hybridization and the sequence analysis Result A total of 22 different fragments ranging from 250 to 750bp 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 of microarray in detecting HCV 1b RNA were high Conclusion The method of preparing microarray probes by construction of cDNA fragments library was effective, quick and simple,the optimized microarray was sensitive and effective in clinical diagnosis of HCV 1b