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Detection and identification of human papillomavirus by PCR and type-specific reverse hybridization

a technology of reverse hybridization and detection method, which is applied in the field of detection and identification of human papillomavirus by reverse hybridization and type-specific reverse hybridization, can solve the problems of insufficient sensitivity and specificity of diagnosis by detection of hpv anti-bodies, and is not possible to diagnose hpv by culture, etc., and achieves rapid and reliable detection methods.

Inactive Publication Date: 2003-09-04
INNOGENETICS NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] It is an aim of the present invention to provide a rapid and reliable method for detection and / or identification of HPV, possibly present in a biological sample.

Problems solved by technology

Diagnosis of HPV by culture is not possible.
Also diagnosis by detection of HPV anti-bodies appears to be hampered by insufficient sensitivity and specificity.

Method used

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  • Detection and identification of human papillomavirus by PCR and type-specific reverse hybridization
  • Detection and identification of human papillomavirus by PCR and type-specific reverse hybridization
  • Detection and identification of human papillomavirus by PCR and type-specific reverse hybridization

Examples

Experimental program
Comparison scheme
Effect test

example 2

Optimization of PCR Primers from the A, B and C region

[0176] Introduction

example 1

[0177] Example 1 describes the selection of semi-conserved regions in the L1 gene of the HPV genome, that permitted the development of a general PCR system. Degenerated primers were used for universal amplification of HPV sequences from different genotypes.

[0178] The present example describes the optimization of the primers aimed at these regions. Instead of degenerated primers, this study aimed at the development of several distinct and defined forward and reverse primers.

[0179] Materials and Methods

[0180] Alignments of L1 sequences were used to deduce PCR primers from the three regions A, B and C (FIG. 1). Primers were tested by PCR in different combinations on plasmids, containing partial or complete genomic inserts from the genital HPV types 6, 11, 16, 18, 31, 33, 35, 39, 43, 45, 51, 52, 53, 56, 57, 58, 59, 62, 64 and 67 as listed in table 2.

[0181] HPV DNA amplification was performed according to the following protocol. The final PCR volume of 100 .mu.l contained 10 .mu.l of HPV...

example 3

Identification of Different HPV Types by Analysis of a Small PCR Fragment Derived from the L1 region

[0187] Introduction

[0188] Identification of the different HPV genotypes may have great clinical and epidemiological importance. Current classification methods are for instance based on either type-specific PCR or sequence analysis of larger DNA fragments. Therefore, there is a clear need for a simple, rapid and reliable genotyping assay for the different HPV genotypes.

[0189] This assay should preferably be combined with the detection of HPV DNA, aiming at the same genomic region. Therefore, we aimed at the development of a screening assay to detect the presence of HPV DNA in clinical samples, and (in case of a positive screening result) the subsequent use of the same amplimer in a genotyping assay.

[0190] The theoretical requirements for such an assay would be as follows:

[0191] 1. The amplimer should be small, to allow highly sensitive detection and to permit amplification from formali...

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Abstract

A method for detection and / or identification of HPV present in a biological sample comprising amplification of HPV polynucleic acids and of hybridization of said amplified polynucleic acids to a number of probes whereby a short fragment of the L1 gene of HPV is amplified after which, the amplimers are contacted with probes that specifically hybridize to the said short fragment of the L1 gene of at least one HPV type and a diagnostic kit to perform said method and primers and probes used in the said method.

Description

[0001] The present invention relates to the field of detection and identification of Human Papillomavirus (HPV) infections in clinical samples.[0002] Cervical cancer is the second most common malignancy in women, following breast cancer. Carcinoma of the cervix is unique in that it is the first major solid tumor in which HPV DNA is found in virtually all cases and in precursor lesions worldwide.[0003] Nowadays, 74 HPV genotypes have been characterized and are numbered in chronological order of isolation. HPV is epitheliotropic and infects only the skin (cutaneous types) or the mucosa of the respiratory and anogenital tract (mucosal types). Thirty-six of the 74 HPV types are known to infect the uterine cervix. Based on the induced benign, premalignant or malignant lesions, HPV is divided into low-risk (e.g., HPV types 6, 11, 42, 43 and 44) and high-risk types (e.g., types 16, 18, 31, 33 and 45), respectively. The high-risk types account for more than 80% of all invasive cervical canc...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/70
CPCC12Q1/708C12Q2531/113
Inventor VAN DOORN, LEEN-JANQUINT, WIMKLETER, BERNHARDTER SCHEGGET, JAN
Owner INNOGENETICS NV
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