随机引物引导的乙型肝炎病毒DNA探针标记和重复杂交

RANDOM PRIMERED LABELLING OF HBV DNA AND ITS APPLICATION IN REHYBRIDIZATION

随机引物引导的探针标记足一种比切中移位更为有效的探针标记方法,作者应用这种方法制备^(32)P标记的乙型肝炎病毒(HBV)DNA探针。经液闪测定,探什比放射性为lxl0^(9)cpm/ug,(32)P掺入率为71.43％,均比切口移位有关参数上限高出数倍。将这种探针用于尼龙膜载样杂交,在每100cm^(2)的样膜所加探针放射性强度为5 x10^(6)cpm、浓度为20ug/L条件下,放射自显影24小时即可出示清晰结果。并且检出HBV DNA灵敏度达0.1Pg,检测HBSAg和HBCAg双阳性血清,HBVDNA阳性率达87％(26/30)。用这种方法还制备地高辛素标记的HBV DNA探针,并在探针浓度为20 ug/L条件下,对^(32)p探针杂交过的样膜重复杂交。结束检出HBV DNA灵敏度为0.05 Pg,检测HBsAg和HBeAg双阳性血清,HBV DNA阳性率为73％(22/30)。上述结果表明随机引物引导标记可用于制备高比放同位素探针和高敏感性非同位索探针。前者对研究微量基因必不可少,后者对基层单位常规开展检测非常重要。

The method of random primered labelling was used to prepare 32P labelled HBV DNA probe. By liquid scintillation, its specific activity was tested to be 109 cpm/μg, incorporation rate of 12P 71.43%, both were higher than those observed in nick translation. By applying it in 5ng (6×106 cpm) in 5 ml per 100 cm2 membrane to detect HBV DNA spotted on nylon filter, the probe was found to have a sensitivity up to 0.1pg and a positivity rate as high as 87%(26/30 )in both HBsAg and HBeAg carrier sera, after the filter was exposed to X-ray film for 24 hours. Digoxigenin labelled HBV DNA probe was also prepared by this technique and applied in 20μg/L(100 ng in 5 ml per 100 cm2 membrane) to rehybridize HBV DNA detected by 32P labelled probe mention above.It gave a sensitivity up to 0.05Pg and a positivity rate as high as 73%(22/30).