应用Digoxigenin标记探针定量检测血清HBV　DNA

Quantitation of HBV DNA in Serum by Dot Hybridization Applying Probe Labelled with Digoxigenin

本文报道采用非同位素Ｄｉｇｏｘｉｇｅｎｉｎ标记探针定量检测血清ＨＢＶＤＮＡ。显色膜经空气干燥、液体石蜡透明处理后，即可置酶联免疫检测仪上测定各斑点的光密度（ＯＤ）值，波长６３０ｎｍ。结果发现已知含量的ＨＢＶＤＮＡ在２－５００ｐｇ／样点范围内与其相应的ＯＤ值有着良好的线性关系（ｒ＝０．９７０ｙ＝０．００１２ｘ＋０．１０５６）。标本测得ＯＤ值后，便可知其含量。该法重复性试验结果良好，１１份ＨＢＶＤＮＡ阳性的血清经定量测定，其ＨＢＶＤＮＡ含量在２０－１０５ｐｇ／４０μｌ之间。定量检测血清ＨＢＶＤＮＡ不仅可用来评价乙肝治疗的效果，而且也可用于了解血清传染性的强弱。

We previously reported a novel,digoxigenin-labelled HBV DNA probe used for the detection of serum HBV DNA,This method has been further developed for quantitative study.After dot hybridization and the addition of Ap-conjugated antibody and substrates,the colour spots developed on nitrocellulose membrane.We dried the membrane and cleared it with paraffin liquid,then the absorbance of each spot was measured at 630 nm in the optical densitometer for ELISA.A known amount of cloned HBV DNA was used as standard. It was found that the optical density (OD) values were proportional to the amounts of HBV DNA added in a range of 2500pg (y=0.0012x+0.1056,r=0.970).A standard curve was thus established.The amount of HBV DNA in an individual sample could hence be determined once its reciprocal OD value was measured.The concentration of HBV DNA in 11 HBV DNA positive sera detected by this method varied from 20-105 pg/40ul.This method could be used for the study of the infectivity of HBV and the effectiveness of antiviral therapies.