乙二胺修饰法标记生物素化HBVDNA探针及其应用

APPLICATION OF BIOTINYLATED HBV DNA PROBE LABELEDBY A CHEMICAL METHOD

采取将核酸用乙二胺作化学修饰后标记生物素的方法制备了生物素化ＨＢＶＤＮＡ探针．该探针的检测敏感性达到０．２５ｐｇ。用它作斑点杂交检测血清ＨＢＶＤＮＡ，结果４１份ＨＢｓＡｇ和ＨＢｅＡｇ阳性的血清有３６份显示斑点阳性；在９份ＨＢｓＡｇ阳性而ＨＢｅＡｇ阴性的血清中，有２份显示斑点阳性；而在５份ＨＢｓＡｇ阴性的正常人血清中未检出ＨＢＶＤＮＡ。

iotinylated HBV(Hepatitis B virus)DNA probe was prepared by a chemical labelingmethod.The method involves the bisulfite catalyzed transamination reaction of cytisine residuesin nucleic acids with ethylenediamine. Primary amino groups on the cytosine derivatives are thenreacted with biotiny-ε -amidocaproic acid-N-hydroxysuccinimide ester.The probe labeled by thismethod was able to detect 0.25 pg homologotis DNA in dot hybridization,When the probe wasused for clinical assay,HBV DNA was detected in 36 of 41 sera specimens in which both HBsantigen and HBe antigen were positive,and it was also detected in 2 of 9 sera specimens in whichHBs antigen was positive but HBe antigen was negative,The probe did not react with 5 normalsera. The results showed that the biotinylated HBV DNA probe labeled by this method was sen-sitive and specific. Also,the chemical labeling method is less expensive than other methods forthe preparation of biotiny-lated HBV DNA probe. The novel technique for labeling biotihylatedHBV DNA probe could lead to the wider application of hybridization techniques in diagnosis ofHBv infection.