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Human papillomavirus gene chip, preparation and application thereof, assay kit and application thereof

A technology of human papillomavirus and gene chip, applied in the field of gene chip detection of 25 different types of human papillomavirus

Active Publication Date: 2010-01-13
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] In China, there have been applications for related patents on HPV typing chips, such as Liu Yuling (public number: CN1544654, public date: 2004.11.10) to detect HPV6, 11, 16, 31, 33, 35, 45, 39, 51, 52 , 56, 58, 32, 34, 13, 40, 41, 42, 44, 53, 54, 55, 74, 66 and 69, a total of 25 types, but the high-risk type 18.59.73.82 was not detected

Method used

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  • Human papillomavirus gene chip, preparation and application thereof, assay kit and application thereof
  • Human papillomavirus gene chip, preparation and application thereof, assay kit and application thereof
  • Human papillomavirus gene chip, preparation and application thereof, assay kit and application thereof

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Experimental program
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Embodiment 1

[0130] Embodiment one Probe design and preparation

[0131] 1. Sequence acquisition:

[0132] (1) HPV: The entire L1 gene sequence of HPV and the entire L1 gene sequence of its close relatives were downloaded from the GenBank public database.

[0133] (2) The method for obtaining the sequence in the human BGP gene is the same as (1).

[0134] 2. Examples of probe design:

[0135] (1) HPV probe: Import the L1 gene sequences of each type of HPV into Glustal X software, select a representative sequence and perform blastn comparison in the public data NCBI to determine whether it can be used as a specific target and the position of the specific target. Import the sequence into OligoArray2.0 software. The parameters were set as follows: -n 20; -l 30; -L 40; -D 3000; -t 79; -T 90; -s 65 °C; -x 65 °C; -N 2; ; -m GGGGG CCCCC TTTTT AAAAA ; -g 15. Run the program to design probes online.

[0136] 3. Probe synthesis: after the 5' end of the probe sequence in Table 1 was extended...

Embodiment 2

[0146] Embodiment two Primer design and preparation

[0147] 1. Example of primer design:

[0148] (1) HPV L1 Gene Universal Amplification Primer: Import all HPV L1 gene sequences into Glustal X software, select a representative sequence from it and import it into Primer Primer 5.0 software, set the length to 70bp~10bp, G+C% Value 40% to 60%, Hairpin: NONE, Dimer: NONE, False Priming: NONE, Cross Dimer: NONE. And look for the nucleotide sequence region suitable for universal primer design, its characteristics basically meet the following conditions: 1, the constant region should include most of the HPV related to reproductive diseases; 2, this region should contain specific probes that are easy to design 3. The constant regions on both sides of this region can satisfy the design of primers; 4. The amplified products of the designed primers should not be too large, otherwise it will affect the PCR sensitivity. The size of the amplified product of the designed HPV primers ...

Embodiment 3

[0153] Embodiment three Rapid detection of 25 different types of HPV by gene chip and preparation of kit

[0154] 1. Sample handling:

[0155] (1) Centrifuge the collected clinical samples at 15000 g for 10 minutes;

[0156] (2) Discard the supernatant, add 100 μl lysate, mix well, and bathe in water at 100°C for 10 minutes;

[0157] (3) The lysate obtained in the previous step was centrifuged at 15000g for 5 minutes;

[0158] (4) Collect the supernatant, which contains genomic DNA and can be used for detection or stored at -20°C.

[0159] Attachment: lysate formula:

[0160] 50mmolL -1 NaOH

[0161] 10mmolL -1 Tris-HCl (pH8.0)

[0162] 0.5% Tween-20

[0163] 0.5% NP-40

[0164] 0.5mmolL -1 EDTA (pH8.0)

[0165] 5% chelex-100

[0166] 2. Amplify the target sequence: take 3 ul of the supernatant extracted by the above genome extraction method as a template and add it to the PCR reaction mixture. The formula of the PCR reaction mixture is shown in Table 3 below. ...

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Abstract

The invention provides a human papillomavirus gene chip, a preparation method and application thereof, an assay kit and application thereof. The gene chip comprises a solid-phase vector and an oligonucleotide probe fixed on the solid-phase vector, wherein the oligonucleotide probe contains a DNA fragment or a complementary DNA fragment thereof selected from L1 genes of the human papillomavirus or human beta globin gene. The invention also provides a preparation method for the gene chip and an assay kit containing the gene chip. The gene chip and the kit have the characteristics of simple and convenient operation, high flux, high accuracy, strong repeatability and the like, can achieve the aim of detecting 25 different types of human papillomavirus, and can be applied to the clinical test, epidemiology analysis and the like of medical and health departments.

Description

technical field [0001] The invention relates to a gene chip and a detection kit containing the gene chip, in particular to a gene chip for detecting 25 different types of human papillomavirus, a preparation method, an application, a detection kit and an application. Background technique [0002] Papillomavirus (Human Papillomavirus, HPV) is a small non-enveloped double-stranded circular DNA virus. In 1949, Straus et al. first observed virus particles under an electron microscope. HPV particles are spherical, icosahedral symmetry, about 45-55nm in diameter, and the full length of the genome is 7.8-8kb. [0003] The HPV genome has 8 open reading frames (ORFs) and an untranslated regulatory region, including early, late transcribed regions and upstream regulatory regions (URR). The early transcription region (E region) consists of 4500bp, encoding seven early proteins, including E1-E7, which are involved in the functions of DNA replication, transcription, translation regulatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 王磊曹勃阳胡品良
Owner TIANJIN BIOCHIP TECH CO LTD
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