采用E6/E7引物介导的多重PCR技术，通过DNA酶免疫分析检测宫颈病变中的高危和低危HPV基因型。

DNA-EIA to detect high and low risk HPV genotypes in cervical lesions with E6/E7 primer mediated multiplex PCR.

作者信息

Clavel C, Rihet S, Masure M, Chypre C, Boulanger J C, Quereux C, Birembaut P

机构信息

Unité de Biologie Cellulaire, Hôpital de la Maison Blanche, CHU de Reims, France.

出版信息

J Clin Pathol. 1998 Jan;51(1):38-43. doi: 10.1136/jcp.51.1.38.

DOI:10.1136/jcp.51.1.38

PMID:9577370

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC500429/

Abstract

BACKGROUND

Oncogenicity of human papillomavirus (HPV) DNA in premalignant and malignant uterine cervical diseases is mainly induced by E6/E7 open reading frame (ORF). The presence of an oncogenic HPV DNA may be a diagnostic marker for the detection of cytologically negative smears.

AIMS

To evaluate an original polymerase chain reaction enzyme immunoassay (PCR-EIA) for the detection and typing of oncogenic and non-oncogenic HPV types.

METHODS

The test was an original multiplex labelled PCR-EIA for the detection and typing of oncogenic and non-oncogenic HPV using three consensus sequence primers within the oncogenic E6/E7 ORF. One primer was dinitrophenyl (DNP) labelled and the DNP labelled amplimers could be further hybridised with specific biotinylated oligoprobes mixed in only two cocktails: oncogenic (16, 18, 31, 33, 35, 52, and 58) and non-oncogenic (6 and 11) HPV types in only two wells; then biotinylated oligoprobes were deposited in streptavidin-coated microplates. The PCR-EIA was validated on HPV plasmids (types 6, 11, 16, 18, 31, 35, 52, and 58) and used to evaluate cervical scrapes from 181 patients (median age 32 years) at high risk for cervical cancer.

RESULTS

HPV were detected in the cervical scrapes of 88 of 181 patients (48.6%); nine with non-oncogenic HPV (5.0%) and 79 with oncogenic HPV (43.6%) including 29 coinfections with oncogenic and non-oncogenic HPV. The number of oncogenic HPV infections increased with the presence of high grade lesions: 95.8% of the cervical scrapes from patients with high grade lesions contained oncogenic HPV compared with 32.1% of the specimens from patients without any lesions detectable by colposcopy and/or by cytological examination of the cervical smears. Moreover, 60% of cervical scrapes exhibiting low grade lesions contained oncogenic HPV.

CONCLUSIONS

This test is simple, specific, sensitive, safe, fast, reproducible, and easy to use in routine practice. Thus, it is possible to detect simultaneously on a simple cervical scrape, two kinds of HPV--oncogenic and non-oncogenic--in just two microplate wells with non-isotopic oligoprobes.

摘要

背景

人乳头瘤病毒（HPV）DNA在子宫颈癌前病变和恶性病变中的致癌性主要由E6/E7开放阅读框（ORF）诱导。致癌性HPV DNA的存在可能是检测细胞学阴性涂片的诊断标志物。

目的

评估一种用于检测致癌性和非致癌性HPV类型并进行分型的新型聚合酶链反应酶免疫测定法（PCR-EIA）。

方法

该检测是一种新型多重标记PCR-EIA，使用致癌性E6/E7 ORF内的三种共有序列引物来检测致癌性和非致癌性HPV并进行分型。一种引物用二硝基苯基（DNP）标记，DNP标记的扩增子可进一步与仅混合在两种混合物中的特异性生物素化寡核苷酸探针杂交：致癌性（16、18、31、33、35、52和58型）和非致癌性（6和11型）HPV类型，仅在两个孔中进行；然后将生物素化寡核苷酸探针沉积在链霉亲和素包被的微孔板中。PCR-EIA在HPV质粒（6、11、16、18、31、35、52和58型）上进行了验证，并用于评估181例宫颈癌高危患者（中位年龄32岁）的宫颈刮片。

结果

181例患者中有88例（48.6%）的宫颈刮片中检测到HPV；9例为非致癌性HPV（5.0%），79例为致癌性HPV（43.6%），其中29例为致癌性和非致癌性HPV合并感染。致癌性HPV感染的数量随着高级别病变的出现而增加：高级别病变患者宫颈刮片中95.8%含有致癌性HPV，而在阴道镜检查和/或宫颈涂片细胞学检查未发现任何病变的患者标本中这一比例为32.1%。此外，表现为低级别病变的宫颈刮片中60%含有致癌性HPV。