病毒性肝炎联合诊断cDNA芯片的研制与临床应用性研究  

Preparation and clinical application of cDNA microarray for combined detection of hepatitis virus

作　　者：孙朝晖[1] 王蜀燕[1] 危敏[2] 石玉玲[1] 李林海[1] 王从容[3] 

机构地区：[1]广州军区广州总医院检验科,广州510010 [2]南方医科大学基因工程研究所 [3]南方医院检验科

出　　处：《解放军医学杂志》2007年第11期1179-1183,共5页Medical Journal of Chinese People's Liberation Army

基　　金：广东省自然科学基金资助项目(7000065);中国博士后科学基金资助项目(20070410831);广东省医学科研基金资助项目(A2007477)

摘　　要：目的制备病毒性肝炎联合诊断cDNA芯片,并进行系列优化及临床应用性研究。方法对于基因组较小的HBV、HDV病毒,采用PrimerPremier5.0软件针对其保守区域设计特异引物,普通PCR技术制备探针;利用限制性显示PCR(RD-PCR)技术制备基因组较大且复杂的HCV芯片探针。为了便于摸索实验条件和更好地进行探针优化,先后制备3种芯片,包括HBV与HDV诊断芯片、HCV诊断芯片及HBV、HDV、HCV联合诊断芯片。结果杂交结果显示HBV与HDV诊断芯片、HCV诊断芯片的诊断效果较为满意。从以上2种芯片中选取探针制备HBV、HDV、HCV联合诊断芯片,并对该芯片的检测质量进行初步评估:①检测线性:病毒质粒拷贝数在104～1011copies/ml之间时,芯片检测法显示了较好的线性(r分别为0.9902、0.9921、0.9819,P<0.01);②特异性:检测黄热病病毒(YFV)、乙型脑炎病毒(JEV)、登革热病毒(DV)等其他病毒样品结果均为阴性,说明该芯片特异性较好;③重复性:检测HBV、HDV、HCV的批内精密度变异系数(Cv)值为7.1%、7.2%、6.6%,批间精密度Cv值为7.9%、8.2%、7.6%;④准确性:将HBV、HDVPCR片段(共14条)、HCV限制性片段(24条)和部分临床阳性血清(PCR产物)全部进行序列测定,在GenBank中进行Blast比较,结果表明克隆基因均属于相应基因,与理论一致。为了适应临床诊断的要求,对实验条件进行优化,包括减少杂交时间、取消预杂交、将杂交温度提高到52℃等措施。在实验室对芯片进行初步评估后,对临床血清标本进行小规模批量检测实验:用芯片法和实时定量PCR法检测HBV阳性血清(98例)、HCV阳性血清(42例)和阴性血清(130例),对两种方法进行对比,结果显示芯片法检测与实时定量PCR法有良好的相关性(HBV、HCV的r值分别为0.9854和0.9582),检测HBV、HCV、HDV的敏感性和特异性分别为85.7%、76.2%、80.0%和96.7%、95.0%、100%。结论本研究研制的联合诊断芯片敏感性、特异性Objective To develop the cDNA microarray for the combined detection of hepatitis virus, and to study the feasibility of applying the rnicroarray in clinical setting. Methods For the small and simple genome of HBV and HDV, the specific primers of PCR were designed with Primer Premier 5.0 program according to the conserved region of HBV and HDV, and 10 and 4 gene fragments were obtained respectively, which could be used As the probes of gene chip. As for the complex genome of HCV, the technique of restriction display PCR （RD-PCR） was employed. Some of gene fragments were selected which were comparatively more specific and sensitive as mi- croarray probes. In order to explore the experimental conditions of microarray in vitro detection, three types of gene chip were prepared successively including HBV and HDV simultaneous detection, HCV detection and modified HCV detection. Results The hybridized signals on the gene chip showed that the effect in detection was satisfactory. Through the prepared gene chips mentioned above, some probes with good quality were selected and the microarray was prepared for HBV, HCV and HDV simultaneous detection. The diagnostic capability of the microarray was evaluated following the washing and scanning steps. Linearity： Serial dilutions of the target DNA or cDNA showed that a strong linear relationship existed between the various concentrations of target DNA or cDNA and the fluorescence intensities obtained from microarray assay （r=0. 990 2, r=0. 992 1, r=0. 981 9）, and that the detection range for the microarray was from 10^1 to 10^11 copies/ml. Specificity： Samples from other viruses such as YFV, JET and DV were also subjected to the test and the results were all negative. Reproducibility： The reproducibility of this assay system was evaluated by repeated measurements, and the within-run coefficient of validation of HBV, HCV and HDV were 7. 1%, 7. 2% and 6. 6%, respectively, while the between-run coefficient of validation was 7.9%, 8. 2% and 7. 6%, respectively. Accuracy

关 键 词：肝炎 病毒性 人 寡核苷酸序列分析 核酸杂交 

分 类 号：R394.2[医药卫生—医学遗传学]