Amplification-hybridisation method for detecting and typing human papillomavirus

一种人乳头瘤病毒、扩增子的技术，应用在属特异和基因型特异探针的序列领域，能够解决昂贵、费时、不能确定基因型等问题

Inactive Publication Date: 2005-07-27
GENOID KFT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in practice, it is not widely used because it is expensive and time-consuming, its use is not acceptable in routine diagnostic laboratories, and in the case of mixed samples, no genotype can be determined

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Synthetic oligonucleotide primers

[0060] The oligonucleotide primers used in the method of the present invention were of commercial origin (IDT, USA). The synthesized primer has a 5' amino group. Use NHS-esters for biotin, fluorescein and digitonin labeling. Labeled nucleotides were purified by HPLC.

Embodiment 2

[0062] Sample processing, DNA preparation

[0063] The gynecologist took out the sample with a cell brush and transferred it into PBS solution (10 mM phosphate buffer solution pH=7.4, Sigma, NaCl 138 mM, KCl 2.7 mM). Samples are pretreated in sample tubes. Before lysing, centrifuge (2000g, 10 minutes) the sample, discard the supernatant, and add 1ml of PBS solution, vortex, centrifuge again, discard the supernatant. At the end of the treatment, 250 μL of lysis buffer (0.5 mg / ml proteinase K, 0.01M TRIS-HCl pH=8, 0.001M EDTA pH=8 in distilled water) containing the internal control for the HPV assay ( SEQ.ID.NO:68), vortex, and incubate at 56°C for 30 minutes. Thereafter, further liquid processing was carried out on the TECAN RSP150 remote control instrument: 200 μL of binding solution (5.5M GUSCN, 20 mM EDTA, 10 mM TRIS-HCl pH=6.5, 65 mM dithiothreitol, 40 g / L silica, SIGMA Cat. No.: 28,851-3, distilled water), and separate silica from soluble components by column filtrati...

Embodiment 3

[0065] Overview of HPV Testing

[0066] amplify

[0067] The total reaction volume was 25 μl, including the following components: 10 μl DNA, 2.5 μl 10×polymerase buffer (final concentration: 10 mM TRIS-HCl (pH=9.0), 50 mM KCl, 0.1% TritonX-100 (Promega), 2 mM MgCl 2 ), 250 μM each dNTP (ATP, CTP, GTP, TTP), 4 μM primer mix: SEQ.ID.NO: 35, 37-40, 73-75 and 1 U Taq DNA polymerase (Promega). The reaction was performed on a GeneAmp 9700 PCR thermal cycler with the following parameters:

[0068] Cycle 1: 95°C for 4 minutes;

[0069] Cycle 2-40: 94°C for 30 seconds, 48°C for 1 minute and 72°C for 45 seconds;

[0070] Cycle 41: 72°C for 3 minutes.

[0071] hybridization and detection

[0072] Hybridization is performed on a solid phase. 24 hours ago, 96-well polystyrene plates (Costar) were coated with streptavidin (0.02 mg / ml streptavidin in PBS solution). Plates were incubated at room temperature and after 24 hours washed with 250 μl (25 mM TRIS pH=7.5, 125 mM NaCl, 2...

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PUM

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Abstract

The present invention provides amplification and hybridisation method for detecting and typing human papillomavirus (HPV), and the primers and hybridisation probes used in the method. The invention relates to a concrete part of the HPV genome, which is suitable for designing HPV genus-specific and HPV genotype-specific hybridisation oligonucleotide probes.

Description

field of invention [0001] The present invention provides improved methods for detecting and typing human papillomaviruses (HPV). [0002] One aspect of the invention defines regions of the HPV genome suitable for the design of HPV genus-specific and HPV genotype-specific hybridization oligonucleotide probes, advantageously adjacent to each other and in one amplicon. [0003] Another aspect of the invention relates to the sequences of genus-specific and genotype-specific probes. [0004] Another aspect of the present invention relates to an optimized method for amplifying HPV genotypes and detecting HPV genotype amplified products and optimized preparation of reagents (kit). Background of the invention [0005] Many papillomavirus sequences have been determined, see the publications cited here as reference: HPV-6: de Villiers et al., J. Virology, 40 (1981); HPV-11: Dartmann et al., Virology 151 , 124-130 (1986); HPV-16: Seedorf et al., Virology 145 , 181-185 (1985); HPV-18...

Claims

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Application Information

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Patent Type & Authority Applications(China)
CPCC12Q1/708
Inventor C·耶奈伊T·陶卡奇
Owner GENOID KFT
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