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HBV YMDD mutation, precore region mutation/BCP region mutation and genotyping integrated-detection DNA microarray chip

A technology of microarray chip and mutation detection, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., and can solve complicated, costly and time-consuming problems

Inactive Publication Date: 2010-03-03
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, domestic manufacturers produce and sell hepatitis B virus resistance mutation detection reagents, but there are no reagents for hepatitis B virus pre-C / BCP mutation and genotyping detection reagents; due to technical limitations, there are no HBV YMDD mutations, pre- The integrated reagents for C region / BCP region mutation and genotyping detection can be completed at one time, often requiring multiple kits for multiple detections, which is complicated and time-consuming

Method used

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  • HBV YMDD mutation, precore region mutation/BCP region mutation and genotyping integrated-detection DNA microarray chip
  • HBV YMDD mutation, precore region mutation/BCP region mutation and genotyping integrated-detection DNA microarray chip
  • HBV YMDD mutation, precore region mutation/BCP region mutation and genotyping integrated-detection DNA microarray chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Design of DNA microarray chip PCR primers and probes for integrated detection of HBV YMDD mutation, pre-C region / BCP mutation and genotyping

[0037] (1) HBV typing: design one upstream primer, two downstream primers, and the downstream primer CY3 fluorescent label, adopt asymmetric PCR amplification system; design 15 specific probes for HBV A, B, C, D typing, These four types are covered.

[0038] (2) HBVYMDD mutation: In order to specifically and efficiently amplify the target gene in the nucleic acid sample to be tested, the whole genome DNA sequence of hepatitis B virus A to H was retrieved from the Gene Bank, targeting the reverse transcriptase gene of hepatitis B virus For sites that are prone to mutation under the selection pressure of nucleotide analogue drugs, use primerpremier5.0 and oligo6.0 software to design two forward primers, one reverse primer, and reverse primer CY3 fluorescent label, and use asymmetric PCR reaction system to amplify the hy...

Embodiment 2

[0040] Example 2: Preparation of DNA microarray chip for integrated detection of HBV YMDD mutation, pre-C region / BCP region mutation and genotyping

[0041] Using a gene chip automatic spotting instrument, 42 detection probes and 6 control probes (the control probes are positive and negative control probes designed for the three detection target conservative regions respectively) were spotted on specific areas of the slide. area to make a DNA microarray.

[0042] Spotting probe solution: dilute the probe into 5×SSC, 0.05% SDS solution, the final concentration is 30 μM;

[0043] Spotting matrix: (see attached figure 1 ) spot the probes on the glass slide, and repeat 2 points of each probe horizontally in the hybridization area, 12 points in each row, 8 rows in total, 96 points; the hybridization area is divided into 10 areas, and 8 samples can be detected at the same time (9,10 hybridization area is the control area).

Embodiment 3

[0044] Embodiment 3: Using the product of the present invention to detect 8 clinical samples

[0045] Take 8 samples and 2 controls, extract viral DNA, first perform DNA sequencing, determine HBV typing, drug resistance and pre-C region / BCP region mutation results, and then use the product of the present invention to detect and verify.

[0046] The DNA microarray chip of the present invention is used to perform one-time detection on the above 8 samples. Take 10 tubes of multiplex PCR reaction solution, add 10 tubes of extracted DNA respectively, 5ul / tube, the total reaction system is 50ul, the PCR cycle parameters are: 94°C 5'; 94°C 30", 55°C 30", 72°C 30", 45 cycles; 72°C 5'.

[0047] Take a microarray chip, add 2ul of 10 tubes of PCR products to the 10 wells of the chip, then add 8μl of hybridization solution (5×SSC) to each well, put the chip into the tank of the automatic hybridization washing machine, and set the hybridization The temperature was 50°C, and the hybridiza...

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Abstract

The invention relates to a DNA microarray chip, in particular to an HBV YMDD mutation, precore region mutation / BCP region mutation and genotyping integrated-detection DNA microarray genetic chip. A solid-phase carrier substrate is used, and an oligonucleotide probe is designed in view of the HBV YMDD mutation, the precore region / BCP region mutation sites and the HBV genotype so as to prepare the DNA microarray chip. The DNA microarray chip can simultaneously detect three indexes at one time and can simultaneously detect a plurality of samples. The DNA microarray chip can be widely used to individual medicine taking guidance in the clinical process of treating HBV infections.

Description

technical field [0001] The invention relates to a DNA microarray chip, in particular to a DNA microarray gene chip for integrated detection of HBV YMDD mutation, pre-C region / BCP region mutation and genotyping. The present invention uses a solid-phase carrier substrate to design oligonucleotide probes for HBVYMDD mutations, pre-C region / BCP region mutation sites, and HBV genotypes, and prepare them into DNA microarray chips, which can simultaneously detect three indicators at one time, And it can detect multiple samples at the same time, and will be widely used in the guidance of individualized medication in the clinical treatment of HBV infection. Background technique [0002] Hepatitis B is an infectious disease caused by hepatitis B virus (HBV), mainly characterized by liver inflammatory lesions and can cause multiple organ damage. There are 2 billion people in the world who have been infected with HBV. At present, there are at least 380 million patients with chronic HBV...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 张帆胡守旺李明何蕴韶
Owner DAAN GENE CO LTD
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