快速反相杂交法检测HBV DNA

A rapid reverse phase hybridization method for detecting HBV DNA in PCR amplified product.

为建立一种简便快速的核酸探针杂交法检测 Ｐ Ｃ Ｒ扩增产物中的 Ｈ Ｂ Ｖ Ｄ Ｎ Ａ，将 Ｐ Ｃ Ｒ 上游引物用生物素标记后进行扩增；核酸探针固定于硝酸纤维素膜上，与带有生物素的 Ｐ Ｃ Ｒ 扩增产物杂交，杂交信号用链霉亲和素 酶结合物显色检测。结果表明，该法检测的敏感性为 ５ ｆｇ，杂交时间为４０ 分钟。２００ 例临床标本检测的阳性率（２７５％ ）高于琼脂糖电泳法（２４５％ ）。同时具有操作简便的特点，可作为 Ｐ Ｃ Ｒ扩增产物中 Ｈ Ｂ Ｖ Ｄ Ｎ Ａ 的常规检测方法。

A simple and rapid hybridization method for detecting HBV DNA in PCR amplified product has been deve loped. One of the PCR primer was labeled with biotin, an oligo nucleotide probe was immobilized on a nitrocellulose membrane. With this system, HBV DNA labeled with biotin in the PCR product could be detected by hybridization. The hybridization signal was detected by streptavidin alkaline phosphatase (SA AP) assay. The result showed that the sensitivity of the hybridization was 5fg, the hybridization needs about 40 min.In 200 clinical speci mens detection,the positive rate (27.5%) of HBV DNA by the hybridization was higher than that of PCR EB(24.5%). The new hybridization method was simple and rapid and can be used as an routine method for detecting HBV DNA in PCR amplified prouducts.