非同位素标记探针检测登革病毒RNA的研究

A STUDY ON DETECTING DENGUE VIRUS RNA BY NUCLEIC ACID HYBRIDIZATION USING NONISOTOPE LABELLED DV2-cDNA PROBES

用地高辛配基（Ｄｉｇｏｘｉｇｅｎｉｎ，Ｄｉｇ）、生物素（Ｂｉｏｔｉｎ，Ｂｉｏ）和辣根过氧化物酶（Ｈｏｒｓｅｒａｄｉｓｈｐｅｒｏｘｉｄａｓｅ，ＨＲＰ）分别标记登革病毒２型（Ｄｅｎｇｕｅｖｉｒｕｓｔｙｐｅ２，Ｄｖ２）的ｃＤＮＡ，制备三种非同位素标记探针，与感染ＤＶ２的Ｃ６／３６细胞上清中病毒核酸做斑点杂交。结果表明：三种标记探针均只与ＤＶ２－ＲＮＡ呈强阳性杂交反应，显示ＤＶ２型特异性；Ｄｉｇ－探针最灵敏，可检出ＤＶ２－ＲＮＡ的最小量为０．１ｐｇ，其次为ＨＲＰ－探针（可检出０．３ｐｇ）和Ｂｉｏ－探针（可检出１～３ｐｇ）。用ＰＣＲ技术制备探针并同步标记比其他方法简便、快速，只需少量模板数小时内可获得足够量的标记探针，是值得推广使用的标记探针制备方法。

igoxigenin,biotin and horseradish peroxidase(HRP) were used to prepare nonisotope labelled DV2-cDNAproberespectively. These probes were used to detect DV-RNA extracted from DV2 infected supernatant of C6/36cell. The results of cDNA : RNA dot blot hybridization showed that these nonisotope labelled probes were highlyspecific. The digoxigenin labelled probe was the most sensitive among the three kinds fo probes and the tiny amount(0.1pof DV2-RNA was detectable.The amount of 0.3pg and 1-3pg DV2-RNA can be detected byHRP and bi-otin labelled probes,respectively.Our results suggest that a large amount of labelled probes can be obtaint using a small quantity of templatewith labelling method.The preparation of labelled probes with PCR may have great value in producing labelledprobes.The modified NaI method for extracting DV-RNA was more convenient and much faster.