巢式PCR方法检测非生殖器部位Bowen病HPV DNA

目的 建立一种巢式ＰＣＲ方法，探讨HPV DNA在非生殖器部位Bowen病中的检出率。方法 巢式PCR方法，用5对不同引物包括CN1FR、CN2FR、CN3FR、CN4FR以及CN5FR进行扩增。结果 通过ClustalX软件，将所设计的每对引物与已知HPV亚型的碱基序列逐一相比较，得知改良设计的引物可以使69种HPV亚型得到扩增，包括黏膜型HPV、皮肤型 HPV以及疣状表皮发育不良相关性 HPV；PCR反应体系的敏感度在10-2～10-3拷贝之间。41例非生殖器部位Bowen病的组织标本中，5例HPV DNA阳性，其中高危黏膜型3例（2例HPV16，1例HPV33），皮肤型2例（HPV27和HPV76各1例）。结论 改良的巢式PCR方法具有较高的敏感性、特异性，部分非生殖器部位Ｂowen病发病与黏膜高危型ＨＰＶ感染相关。

Detection of human papillomavirus DNA in extragenital Bowen's disease by using a nested PCR

Objective To develop a new nested PCR to detect human papillomavirus （HPV） DNA in lesions of extragenital Bowen′s disease. Methods DNA was extracted from the lesions of 41 patients with extragenital Bowen′s disease and subjected to the amplification of HPV by a nested PCR. Five primers, including CN1FR, CN2FR, CN3FR, CN4FR and CN5FR, were designed and used in the second round PCR. Results Based on the ClustalX analysis, 69 HPV subtypes, including mucosal types, cutaneous types and epidermodysplasia verruciformis-associated types, could be amplified by using the 5 designed primers. The detection limit varied from 10-3 to 10-2 copies of HPV DNA for this PCR. Of the 41 lesional specimens, 5 were positive for HPV DNA, including 3 cases of high-risk HPV types （2 cases of HPV 16, 1 case of HPV 33） and 2 cases of cutaneous HPV types （1 case of HPV 27 and 1 case of HPV 76）. Conclusions The improved nested PCR is highly sensitive and specific for the detection of HPV DNA in lesions of extragenital Bowen′s disease. The development of extragenital Bowen′s disease may be associated with the infection with high-risk mucosal HPV types in some patients.