利用寡核苷酸微阵列技术对HBV、HCV、HIV、HAV、GBV-C/HGV和B19进行同时监测的初步研究

探讨研制能同时检测HBV、HCV、HIV、HAV、GBV-C/HGV和B19的微阵列监控芯片。根据病毒公开发表序列,序列比对,得出保守区域,设计病毒的特异性检测探针,同时设置阴性、阳性参照探针,制备监控微阵列。利用随机引物PCR方法标记样品中的病毒靶序列,标记产物与微阵列上的探针杂交,清洗、扫描后进行结果分析。通过对质粒或模式分子的检测以及经HBV、HCV、HIV临床标本的验证,发现该微阵列监控芯片具有良好的特异性。其对质粒的检测灵敏度可达102病毒拷贝数,对临床标本的检测灵敏度可达103病毒拷贝数。此外,该微阵列监控芯片可检测出病毒混合感染血清。为微阵列监控芯片应用于此六种血液病毒的检测打下一定的基础。

Simultaneous Detection of Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus Type-1, Hepatitis A Virus, Hepatitis G Virus and Human Parvovirus B19 by Oligonucleotide Microarray Hybridization

We described the diagnosis microarray for simultaneous detection of hepatitis B virus,hepatitis C virus,human immunodeficiency virus type-1,hepatitis A virus,hepatitis G virus and human parvovirus B19.Fully sequenced viral genomes were obtained from GenBank and aligned to find out the most conserved regions on which designed oligonucleotide probes were based.With all the specific probes corresponding to the six types of viruses to be detected in our experiment,diagnosis microarrays were fabricated,in which negative and positive probes were used as control.On the other hand,the tested viral nucleotides were amplified,fluorescently labeled and hybridized with the diagnosis microarray.Then the microarrays were scanned using a GenePix 4100A scanner,and the data obtained were analyzed.The diagnosis microarray showed high degree of specificity and identified successfully the viruses in clinical samples,and could detect efficiently 10~2 copies of the plasmid and 10~3 virus copies in clinical samples.Clinical samples coinfected with several viruses could be detected simultaneously.The assay demonstrated the potential of microarray for diagnosis of HBV,HCV,HIV,HAV,GBV-C/HGV and B19.