摘要
目的 研发检测拉米夫定耐药的基因芯片进行临床拉米夫定致乙型肝炎病毒 (HBV)耐药相关基因突变的监测。方法 ( 1)将HBVYMDD区 4个突变位点为靶的 16条寡核苷酸探针 ,用GMS 417芯片点样仪固定在经特殊处理的玻片上。待检HBV突变相关的核酸经过聚合酶链反应(PCR)扩增 ,及用Cy5标记的三磷酸脱氧胞苷进行荧光标记 ,再通过与基因芯片杂交 ,严谨洗涤 ,将非突变的标记片段洗脱后 ,将芯片在GeneTACLSIV扫描仪下进行扫描 ,计算机解读。 ( 2 )应用PCR定点突变技术构建Leu5 15Met,Met5 39Ile ,Met5 39Val和V5 42I 4位点和 4位点的单一突变质粒 ,并将芯片检测结果与测序结果对照 ,以鉴定其特异性。同时 ,用血清标本和质粒的重复检测 ,测定其重复性 ,并对 5 0份拉米夫定治疗血清和慢性HBV感染患者血清进行检测。结果 我们研制的芯片能同时特异性地检测耐药相关单一或多位点突变。其检测与测序的符合率 98%。重复率达 96 %～ 10 0 %。结论本研究制作的 4位点基因芯片 ,既可检测乙型肝炎拉米夫定耐药相关单一碱基突变 ,亦可一次有效检出 4个位点的突变 ,且具有快速、高特异性和可重复性 ,检测结果可靠的优点。
Objective To study the development a clinical useful microarray which can detect 4 most commonly and well documented sites for detection of lamivudine-resistance mutants in HBV lamivudine therapy. Methods (1) The selected 16 oligonucleotide probes to screen 4 mutated sites in YMDD region are immoblized in a specific treatment slice by GMS 417 Arrayer. The target 308 bp nucleic acids segment of HBV were amplificated and labeled with Cy5-dCTP fluorescent dye by PCR. After hybridization of target DNA with microarray, the microchips were scanned with Gene TAC LS IV scanner and the data were obtained after processed in computer. (2) To evaluated the specialty and reproducibility of the microarray, the plasmid with Leu515Met, Met539Ile, Met539Val,V542I mutations were condstructed and serve as positive sample. Another 50 sera of lamivudine treated Hepatitis B patients for 6 to 12 months as well as 50 sera without lamivudine administration were assayed by this microarray to evaluated the rate and genetype in lamivudine related resistance mutation. Results The microarray can identify a single base change of selected lamivudine resistance-related mutation and multiple mutation detection by a single assay as well. The specialty are well in agreement with sequencing for 98%. The reproducibility rate of repeat examination is range from 96%-100%. Conclusion The microarray of lamivuine resistance related mutation can identify a single base change as multiple mutations well. And its hight reproducible results may be useful for clinical monitoring lamivudine resistance related HBV mutation.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2003年第2期83-85,共3页
Chinese Journal of Laboratory Medicine
