肛门生殖器区肿瘤组织中HPV的快速检测和分型

RAPID DETECTION AND GENOTYPING OF HUMAN PAPILLOMAVIRUS IN THE TUMOR TISSUE OF THE ANO-GENITAL REGION

迄今已发现的人乳头瘤病毒（ＨＰＶ） 型已超过７７ 型，其中至少有３０ 个型与泌尿生殖道肿瘤有关。所以ＨＰＶ 的检测与分型对于泌尿生殖道肿瘤的病因和预后将是非常重要的。ＨＰＶ有很大的异质性，故用常规的诊断技术来测定未知标本中的ＨＰＶ 基因型有一定困难。为此，我们应用了反向点杂交法（ＲＤＢ） 来检测ＨＰＶ 分型。即用７ 种序列特异性寡核苷酸探针分别对应７ 型ＨＰＶ（ＨＰＶ６Ｂ、１１ 、１６ 、１８ 、３１ 、３３ 、３５） ，这些探针被合成后依次固定在一条尼龙膜上形成７ 个点，再与样品ＤＮＡ序列的ＰＣＲ产物进行杂交。结果７ 型ＨＰＶ 中任一型阳性都可以在一条尼龙膜上鉴别出来。我们收集来自广州各大医院的标本，共计１７７ 例（ 除２０ 例食道癌标本来自河南省外） ，用ＲＤＢ来检测ＨＰＶ 和分型。结果如下：子宫颈癌８６ 例，其中鳞癌６８ 例，ＨＰＶ 阳性５９ 例（８６ ．７６ ％ ） ；腺癌１８ 例，ＨＰＶ 阳性８ 例（４４ ．４４ ％ ）；尖锐湿疣４２ 例，ＨＰＶ 阳性３９ 例（９２ ．８６ ％ ） ；慢性宫颈炎２９例，ＨＰＶ阳性１ 例（３．４５ ％ ） ；食道癌２０ 例，ＨＰＶ 阳性７ 例（３５ ．００ ％ ） 。

More than 77 types of human papillomavirus (HPV) have been found up to now, among which at least 30 types are associated with uro-genital tumors. So the detection and genotyping would be of great importance for studying the etiology and prognosis of the uro-genital tumors. However, HPV has great heterogeneity, so the detection of HPV type in an unknown specimen with routine diagnostic techniques would be somewhat difficult. Hence we used the reverse dot blot (RDB) technique to detect and genpotype the infecting HPVs. Seven sequence-specific oligonucleotide probes, corresponding respectively to 7 types of HPV(HPV6B, 11,16,18,31,33,,and 35) were used. After the synthesis of these 7 types of probes, they were fixed successively in order on a strip of nylon membrane to form 7 spots, then they were hybridized with the PCR products of the specimen DNA sequences. The result is that any one of the 7 types of HPV DNA, if positive, can be identified on one nylon strip. We collected 177 specimens from the hospitals in Guangzhou (except that 20 cases of esophageal carcinoma were from Henan Province). RDB technique was used to detect and genotype the infecting HPVs and the results were as follows: Uterine cervical carcinoma 86 cases, including 68 squamous carcinomas and 18 adenocarcinomas, the HPV positive numbers were 59/68 (86.76%) of the former (47 for type 16, 9 for type 18, 3 for type 16, 18 mixed infection) and 8/18 (44.44%) of the latter (5 for type 18, 3 for type 16); condyloma acuminatum 42 cases with 39 HPV positive ones (92.86%) of which 17 were type 6B, 20 of type 11 and 2 of mixed infection of types 6B and 11;chronic cervicitis 29 cases, with only one HPV positive (3.45%); and finally esophageal carcinoma 20 cases, with 7 HPV positive ones (35.00%).