PCR结合酶切分析鉴别疱疹病毒科四种病毒感染研究

【作者】 白雪丽；

【导师】 强新；

【作者基本信息】 青岛大学 ， 病原生物学， 2001， 硕士

【摘要】 用DNAstar软件包分别从HCMV、HSV-1、HSV-2和EBV的DNA聚合酶高保守基因区中设计一对共享序列引物，根据四种基因产物序列选择Sma I 和 BamH I作为限制性内切酶，对PCR产物进行酶切，从而做到在一个方法中，经一次PCR扩增并结合酶切即可区分四种病毒的感染。应用这一方法，分别检测了临床高危感染者血标本、孕期可疑宫颈分泌物和尿配对标本共99例，四种病毒扩增的产物分别是HCMV 594bp、HSV-1与 HSV-2均为522bp、EBV 528bp。HSV-1被SmaI酶切形成478和44 bp两个片段，但没有BamH I酶切位点；HSV-2被BamH I酶切后形成224和298bp两个片段，没有SmaI酶切位点；EBV各有一个SmaI和 BamH 酶切位点，分别切成101与427，246和282bp 四个片段；HCMV没有两种酶切位点。经Southern印记、CC法金标准对比、原位杂交和与单一四种PCR试剂进行了验证和评估，结果表明，单一PCR试剂盒和共享引物PCR酶切两种试剂的检测敏感性无明显的差异(X2 =0.29, P>0.05)，符合率达85.7%。与金标准比较，诊断敏感性、诊断特异性和诊断指数，分别为100%、92.3%和192.3%，均在理想范围内。PCR结果酶切法可作为一种简易，快速，敏感和特异的对四种疱疹病毒科病毒基因鉴别诊断试剂，从而为临床提供诊断参考。此法的建立对早期发现孕妇活动性HSV1或HCMV感染并及时予以特异治疗,防止和减少先天性畸形以及优生优育实验室监测工作有重要意更多还原

【Abstract】 single pair of oligonucleotide primer selected within a highly conserved region of the DNA polymerase gene of the herpesvirus was designed to amplify related viral genomes, i.e. herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalovirus, Epstein-Barr virus, by the polymerase chain reaction. A simple restriction enzyme analysis(with SmaI and BamHI) of these amplified products allowed accurate characterization of the herpesvirus type. Cervical mucus and urine , and blood samples from 99 patients who were suspected with different hepesvirus infections were tested for the presence and identification of herpesvirus DNA by this approach. The amplifed products were visualized on ethidium bromide -stained gel with expected size: HCMV 594bp, HSV1,2 522bp, EBV 528bp. The HSV1 amplified product was cleaved by SmaI into 478bp and 44bp fragments and remained undigested by BamHI; conversely, the HSV2 amplified product was cleaved by BamHI into 224bp and 298bp fragments and remained undigested by SmaI. The EBV amplified product was cleaved by SmaI and BamHI into two fragments 101 and 427bp, 282 and 246bp, respectively. While HCMV remained undigested by both restriction enzymes. The specificity of the PCR products were confirmed by Southern blot using oligonucleotide probes specific for each of the four hepesvirus amplified products before restriction enzyme analysis. This assay was also compared with the conventional cell culture(Gold standard) combined with in situ hybridization and the specific PCR for each of the four herpesviruses, and the agreement of 85.7%, sensitivity of 100%, specificity of 92.3% , and dia-index number of 192.3% were derived. The results show that this polymerase chain reaction provides a highly sensitive and specific technique for the identification of herpesviruses DNA and should be of value for early and rapid diagnosis.更多还原