PCR结合核酸分子杂交放射自显影检测HPV16,18实验条件的研究  

Experimental Condition for Detection of HPV16,18 DNA Using PCR and Dot Blot Autoracliogrophy

作　　者：邓涛[1] 张秋萍[1] 于皆平 罗和生 沈志祥 屈雪菊 

机构地区：[1]基础医学院微生物与免疫学教研室

出　　处：《湖北医科大学学报》1997年第2期104-107,共4页

基　　金：国家自然科学基金

摘　　要：为建立一种快速、简便、高敏感性和特异性的检测消化道肿瘤组织中ＨＰＶ１６，１８ＤＮＡ序列的方法，用ＰＣＲ扩增ＨＰＶ１６早期区Ｅ６的１２０ｂｐ片段和扩增ＨＰＶ１８Ｅ６、Ｅ７区２６７ｂｐ片段，扩增产物与相应的５′－末端Ｐ３２标记的特异性寡核苷酸探针进行斑点杂交放射性自显影。探讨了影响ＰＣＲ和杂交的关键因素，从中选出较佳的反应条件：即ＰＣＲ退火温度及杂交温度均为５５℃，反应缓冲液镁离子浓度为１．５ｍｍｏｌ／Ｌ，引物浓度各为５０ｐｍｏｌ／１００μｌ，探针浓度各为１．０ｐｍｏｌ／ｍｌ，杂交时间３ｈ，放射性自显影时间２４ｈ。在上述条件下，该方法可检出低至０．０１ｆｇ的ＨＰＶ１６或ＨＰＶ１８质粒ＤＮＡ，ＨＰＶ１６与ＨＰＶ１８之间无交叉反应，为进一步探讨ＨＰＶ与消化道肿瘤的关系奠定了基础。In order to establish a rapid, easy, highly sensitive and specific method for detecting and screening HPV16,18 DNA sequences in large number of fresh carcinoma tissues from the digestive tract, 120 bp DNA fragment from HPV16 Early Region E 6 and 267 bp DNA fragment from HPV18E 6、E 7 were amplified by polymerase chain reaction. PCR products were hybridized with the specific probes by means of dot blotting. The optimal reaction conditions were tested. It was proved that the reaction optimization is achieved when PCR annealing or hybridizing temperature is 55 ℃, Mg 2+ concentration 1.5 mmol/L in PCR buffer. The primer concentration each 50 pmol/100 μl per reaction, the probe concentration each 0.5 pmol/100 ml, and the autoradiography time 12～24 h. As few as 0.01 fg plasmid HPV16 or HPV18 DNA were detected in the above conditions. The assay specifically detected HPV16 or HPV18 DNA without cross reactivity. The rapid and sensitive analysis of HPV in the tissues using this technique may contribute significantly to identifying the role of HPV in carcinoma from the digestive tract.

关 键 词：乳头状瘤病毒 放射性自显影术 消化道肿瘤 PCR 

分 类 号：R735.04[医药卫生—肿瘤]