聚合酶链反应标记轮状病毒地高辛素探针的初步应用

Detection of rotavirus RNA using DIG labelled probe prepared by polymerase chain reaction

目的为探讨聚合酶链反应（ＰＣＲ）技术标记的地高辛素（ＤＩＧ）探针的特异性和敏感性。方法用聚合酶链反应技术制备地高辛素标记的人类轮状病毒（ＨＲＶ）ｃＤＮＡ探针，经ｃＤＮＡ－ＲＮＡ斑点杂交。结果该探针具ＨＲＶ特异性，可检出１０ｐｇ的ＲＮＡ。应用该项技术检测了１２０份婴幼儿腹泻粪样标本，其阳性率为６５％，明显高于ＰＡＧＥ方法（４９．１％）的阳性率。结论ＰＣＲ方法直接制备地高辛素标记的ｃＤＮＡ探针方便、快速、特异性好、标记率高。

The digoxigenin (DIG) labelled cDNA probe of rotaviruse was directly prepared by polymerase chain reaction(PCR). The results of cDNA-RNA hybridizaton showed that the DIG-cDNA probe exhibits rotavirus specificity and can detect as tiny as 10pg rotavirus RNA. 120 fecal samples of diarrhea from infants and young children were tested by dot-blot hybridization. It was shown that the positive rate of dot-blot hybridization(65.0%)was significantly higher than that of PAGE(49.1%). This study indicated that direct preparation of DIG-labelled rotavirus-cDNA probe by PCR is much faster and simpler than common method of labeling. 