摘要
目的建立茎环状逆转录引物（简称茎环引物）的SYBR GreenⅠ实时荧光定量聚合酶链反应（FQ-PCR）,并作初步应用。方法应用SYBR GreenⅠ实时FQ-PCR检测CD4＋T细胞株的miR-155,初步验证方法的可行性。用所建立的方法对60份临床宫颈刮取物标本进行检测,分析miR-155与宫颈癌及感染的高危型人类乳头状瘤病毒（HPV）基因型别的关系,其聚合酶链反应（PCR）反应产物通过电泳验证产物特异性。结果所建方法能特异的检测到miR-155的扩增信号,最低检出限为10^-7nmol/L,在10^-1-10^-6nmol/L之间有良好的线性关系,相关系数（r）为0.99,扩增后惟一的熔解曲线特异峰显示很好的特异性。CD4^＋T细胞株及60例临床宫颈刮取物标本miR-155均显示阳性,PCR反应产物电泳可获得单一条带。宫颈刮取物miR-155与宫颈癌有关（P〈0.05）,但与本研究涉及的HPV基因型别无关（P〉0.05）。结论所建立的茎环引物的SYBR GreenⅠ实时FQ-PCR能快速、敏感、特异地检测细胞株和临床宫颈刮取物miR-155的表达水平,为进一步研究miR-155及其临床应用奠定了基础。
Objective To establish a method of SYBR GreenⅠreal-time fluorescence quantitative polymerase chain reaction（FQ-PCR） with stem-loop reverse transcription（RT） primer to detect miR-155 and apply it primarily.Methods miR-155 in CD4^＋T cell was determined by SYBR GreenⅠreal-time FQ-PCR.The feasibility of this method was primarily validated.miR-155 in 60 cell specimens scraped from the cervix was detected.The relation of miR-155 with cervix cancer and HPV genotypes was analyzed.The PCR production was analyzed by agarose gel electrophoresis.Results The results showed that the SYBR GreenⅠFQ-PCR with stem-loop RT primer was highly specific and had a broad linear detecting range（10^-1nmol/L-10^-6 nmol/L,the lowest detecting limit： 10^-7 nmol/L,r=0.99）.The melting curve analysis showed a single peak.The results from CD4^＋ T cell lines and 60 cell specimens scraped from the cervix in this assay showed positive.The PCR product migrated as a pure band during electrophoresis.The expression of miR-155 in cell specimens scraped from the cervix was not related to their HPV genotypes in this study（P〉0.05）,but was related to cervix cancer（P〈0.05）.Conclusions The method of SYBR GreenⅠFQ-PCR with stem-loop RT primer can detect miR-155 rapidly,sensitively and specifically in cell lines and cell specimens scraped from the cervix.It lays a foundation to further study on the miR-155 and its clinical application.
出处
《检验医学》
CAS
北大核心
2010年第4期296-300,共5页
Laboratory Medicine
