HRP标记及ECL检测HCMV患者尿样的斑点杂交法  

A Dot-blot Assay Using HRP Direct-labelling and ECL Detection System in Detecting Human Cytomegalovirus Infection in Patients'Urine Samples

作　　者：周越[1] 徐桂林[1] 陆德源[1] 

机构地区：[1]上海第二医科大学微生物学教研室

出　　处：《上海交通大学学报（医学版）》1995年第S1期50-53,共4页Journal of Shanghai Jiao tong University:Medical Science

摘　　要：探讨用ＨＲＰ直接标记ＨＣＭＶＤＮＡＨｉｎｄⅢＪ、Ｎ、Ｏ片段为探针的斑点杂交及ＥＣＬ的方法检测。通过各种条件的探索，认为预杂交约３ｈ，洗涤液分次洗涤及５～８ｍｉｎ曝光时间可取得理想的杂交信号。在此条件下的实验结果表明：ＨＲＰ直接标记的探针与ＨＳＶ－１、ＨＳＶ－２无交叉反应，加用ＥＣＬ技术后检测灵敏度达０．２ｐｇ，且ＨＲＰ标记及ＥＣＬ检测系统的稳定性达０．２ｐｇ。与病毒分离培养及光敏生物素标记探针对临床尿标本的平行检测结果相比，检出阳性率均无显著性差异。研究表明，ＥＣＬ检测方法是一种快速诊断ＨＣＭＶ感染的方法，有其推广价值。The use of HCMV DNA Hind Ⅲ J, N, O fragments as single strand nucleic acid probe direct-labelled with horseradish peroxidase (HRP) for detection of HCMV patients'urine samples by enhanced chemiluminescence (ECL) is described. According to our study, the optimum condition for a satisfactory hybridization signal is a 3h-prehybridization,high-stringent wash buffer after hybridization and 5-8min exposure time. Probe labelled with HRP does not cross-hybridize with HSV-1 and HSV-2DNA, and the whole system is stable in more than 6m.Meanwhile its sensitivity remains 0.2 pg. A parallel study of urine samples of 42 women who had abnormal pregnant history using ECL, probe labelled with photo-biotin, and virus isolation culture showed no significant difference in HCMV DNA positive rate.Therefore, HRP direct-labelling and ECL detection system is the newest assay for rapid diagnosis of HCMV infection, which is worthy of popularizing in both clinical and laboratory applications.

关 键 词：巨细胞病毒 斑点杂交 辣根过氧化物酶 

分 类 号：R446.122[医药卫生—诊断学]