地高辛素标记乙型肝炎病毒DNA探针的制备及应用

PREPARATION AND APPLICATION OF DIGOXIGENIN- LABELLED HBV DNA PROBE

采用随机引物法以地高辛素标记的脱氧尿嘧啶核苷三磷酸(dig-II-dUTP)标记克隆的乙型肝炎病毒(HBV)DNA adw/pAM6,制得高特异性和高敏感性的HBV DNA探针。用于斑点杂交检测HBV DNA,最大敏感度为0.lpg。用于检测HBsAg阳性血清,HBV DNA阳性率为82.7％(91/110),与市售同类药盒相比,阴阳结果一致,与缺口翻译的生物索探针相比,检出率有显著提高(P<0.01)。用于肝癌组织与血清HBV DNA研究,与既往用~32P探针所得结果相符。该探针操作简单、使用方便、出结果快速、价格便宜又无需特殊设备,故适于在临床单位尤其是基层医疗单位应用推广。

Bam H I cut HBV DNA ( adw ) from pAM6 was labelled with digoxigenin-11-dUTP by method of random primers labelling. In a modified reaction when 1 μg DNA was incubated at 37℃ for 20 hours, 0.4 μg probe yielded with high sensitivity and specifity. By dot-blot hybridization, the probe can detect as less as 0.1 Pg HBV DNA, which could satisfy the need of laboratory research or clinical routine use. When it was applied to a detection of HBV DNA from 110 HBsAg positive sera, its pick-up value was as high as 82.7%. By comparison with commercially available digoxigenin-labelled kit, both probes gave a similar positivity of 74/91 ( 81.3% ) . In another comparison when nick-translated Biotin-HBV DNA was employed, the digoxigenin-labelled probe gave a significantly higher positive result (P<0.01). Furthur application in an analysis of some sera and tumor tissue from hepatocellular carcinomas, it showed that Digoxigenin-labelled probe gave a similar positivity to that of 32P-labelled probe reported previously. It becomes clear, however, the digoxigenin-labelled HBV DNA probe is worthy of being explored in China, because of its low price, simple procedure, easy to store, and time saving.