一种基于荧光偏振的人乳头瘤病毒基因型高通量检测方法。

A high throughout assay for human papillomavirus genotypes with fluorescence polarization.

作者信息

Zhang Ju, Yan Xiaojun, Sun Jianzhong, Chen Zhongcan, Gao Yan'e, Bai Yujie, Liu Zhiguang

机构信息

Institute of Gene Diagnosis, Fourth Military Medical University, Xi'an 710004, China.

出版信息

Chin Med J (Engl). 2003 Aug;116(8):1137-40.

PMID:12935397

Abstract

OBJECTIVE

To develop a simple, cheap, quick, accurate and practical method for a high throughout genotypes assay of human papillomavirus (HPV) DNA.

METHODS

Crude DNA was extracted by a simplified proteinase K digesting method. HPV common conservative primers: GP5+/6+ system was used to amplify HPV DNA in 127 samples of condylomata acuminatum (CA) and cervical scrapes by PCR, then the PCR product was assayed using a template directing terminator incorporation (TDI) and genotypes were detected with fluorescence polarization (FP). Major HPVs type-specific probes (HPV6, 11, 16, 18, 31, 33, 35 and 58) designed by us were hybridized with the specific PCR products and a special fluorescent ddNTP terminator was directly added to the end of the probe under direction of specific PCR products. The results were measured with FP and compared with the results of the DNA sequence.

RESULTS

Compared with the results of DNA sequencing, the results detected with fluorescence polarization were all correct. The proposed method could detect more than one type of HPV infection, but DNA sequencing method could not. The positive rate of HPV was 100% in 78 CA biopsies. Among them, there were 14 HPV double infections [HPV6B and 11 (9 cases), HPV11 and 16 (4), HPV11 and 18 (1)], 5 HPV triple infections [HPV6B, 11 and 16 (4), HPV11, 16 and 18 (1)], and one HPV quadruple infection (HPV6B, 11, 16 and 18). The positive rate of HPV was 77% in the 49 cervical scrapes. Six HPV double infections [HPV6B and 11 (2), HPV11 and 16 (1), HPV6B and 16 (1), HPV16 and 18 (1), HPV18 and 58 (1)], 3 HPV triple infections [HPV6B, 11 and 16 (2), HPV11, 16 and 18 (1)] and one HPV quadruple infection (HPV6B, 11, 16 and 18) were detected in cervical cancer scrapes.

CONCLUSIONS

The proposed method allowed a high throughout, special, simple, rapid, automatic and economical detection of HPV-DNA genotyping without a use of labeling probes. It can detect multiple HPV genotype infection and will be and useful tool in HPV genotype screening.

摘要

目的

开发一种简单、廉价、快速、准确且实用的方法，用于高通量检测人乳头瘤病毒（HPV）DNA的基因型。

方法

采用简化的蛋白酶K消化法提取粗DNA。HPV通用保守引物：GP5+/6+系统用于通过PCR扩增127例尖锐湿疣（CA）和宫颈刮片样本中的HPV DNA，然后使用模板导向终止子掺入（TDI）法检测PCR产物，并通过荧光偏振（FP）检测基因型。我们设计的主要HPV型特异性探针（HPV6、11、16、18、31、33、35和58）与特异性PCR产物杂交，并在特异性PCR产物的引导下将一种特殊的荧光双脱氧核苷酸三磷酸终止子直接添加到探针末端。用FP测量结果并与DNA序列结果进行比较。

结果

与DNA测序结果相比，荧光偏振检测结果全部正确。所提出的方法能够检测出一种以上类型的HPV感染，但DNA测序方法则不能。78例CA活检样本中HPV阳性率为100%。其中，有14例HPV双重感染[HPV6B和11（9例），HPV11和16（4例），HPV11和18（1例）]，5例HPV三重感染[HPV6B、11和16（4例），HPV11、16和18（1例）]，以及1例HPV四重感染（HPV6B、11、16和18）。49例宫颈刮片样本中HPV阳性率为77%。在宫颈癌刮片中检测到6例HPV双重感染[HPV6B和11（2例），HPV11和16（1例），HPV6B和16（1例），HPV16和18（1例），HPV18和58（1例）]，3例HPV三重感染[HPV6B、11和16（2例），HPV11、16和18（1例）]以及1例HPV四重感染（HPV6B、11、16和18）。