Peptide nucleic acid chip for detecting muatatonal site of hepatitis B virus and its preparing method

A technology of hepatitis B virus and mutation sites, applied in the field of gene chips

Inactive Publication Date: 2006-01-11
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention uses labeled RNA and peptide nucleic acid chip hybridization to detect hepatitis B mutation sites, which can improve the sensitivity and specificity of detection, which has not been reported so far

Method used

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  • Peptide nucleic acid chip for detecting muatatonal site of hepatitis B virus and its preparing method
  • Peptide nucleic acid chip for detecting muatatonal site of hepatitis B virus and its preparing method
  • Peptide nucleic acid chip for detecting muatatonal site of hepatitis B virus and its preparing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: The structure is attached figure 1 Shown:

[0066] Peptide nucleic acid probes and control dot coating 2 are uniformly distributed on the substrate 1 in an array, which contains 24 detection probes with known mutation sites and wild sites in the pre-hepatitis C and S regions, and 2 are positive Control probe, 1 negative control probe and blank spot liquid control. The positive probes are located around the array. It is not only a positive control, but also a positive signal to locate the array. The PCR amplified product of hepatitis B DNA sample is transcribed and labeled in vitro and hybridized with the peptide nucleic acid array, and the result is obtained by scanning with a laser confocal scanner, and then analyzed and judged.

Embodiment 2

[0067] Example 2: Modification of glass slides and immobilization of peptide nucleic acid probes

[0068] 1. Modification of slides

[0069] The blank glass slide was washed with 10% NaOH, then washed with 1% HCI acid solution, washed with water and then dried to obtain a clean glass bare chip. Treated with 1% 3-aminopropyltrimethoxysilane in 95% acetone solution for 30 minutes, and then washed with acetone 3 times for 5 minutes each time, and baked at 80°C for 15 minutes to obtain the aminated glass slide. Continue to treat with 10% glutaraldehyde aqueous solution for 30 minutes, then wash with double distilled water, and dry in vacuo to obtain an aldehyde modified glass slide.

[0070] 2. Fixation of peptide nucleic acid detection probe (taking aldehyde-based glass slide as an example)

[0071] The design and selection of peptide nucleic acid detection probes are shown in Table 1.

[0072] Fixation of peptide nucleic acid detection probes on aldehyde-based glass slides, using sa...

Embodiment 3

[0079] Example 3: DNA extraction, PCR amplification and labeling of hepatitis B samples

[0080] 1. DNA extraction of hepatitis B samples

[0081] 1) Take 100 μl of hepatitis B serum, add 3 times the volume of lysis buffer (4M guanidine thiocyanate, mercaptoethanol, 0.1 M Tris-CI, pH 7.5), and mix.

[0082] 2) Add an equal volume of chloroform and isoamyl alcohol solution (24:1) and mix well. Centrifuge at 12000 rpm for 15 minutes at room temperature.

[0083] 3) Carefully transfer the supernatant to another 1.5ml centrifuge tube, and add 2 times the volume of absolute ethanol. Place at -20°C for 2 hours. Centrifuge at 12000rpm for 15 minutes at 4°C.

[0084] 4) The precipitate obtained after centrifugation was washed twice with 70% ethanol, and centrifuged at 12000 rpm for 10 minutes at 4°C each time.

[0085] 5) The precipitate is naturally dried and dissolved in 16 μl pure water.

[0086] 2. Amplification and purification of PCR target fragments

[0087] 1) The PCR amplificatio...

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Abstract

A peptide nucleic acid chip for detecting the mutation sites of hepatitis B virus is composed of glass substrate or nylon film, peptide nucleic acid probes array and reference point-type coated layer. Its preparing process includes PCR amplification of hepatitis B virus DNA speciment, fluorescence labelling, and hybridization. The mutation sites can be determined according to the intensity of hybrid signals. Its advantages are high speed, high effect, and high correctness.

Description

1. Technical Field [0001] The invention relates to the field of gene chips, in particular to a peptide nucleic acid low-density chip and a preparation method thereof, in particular to a peptide nucleic acid detection chip of hepatitis B mutation site and a preparation method thereof. 2. Background technology [0002] Gene chip technology was first developed based on the demand for high-throughput and parallel analysis. It is mainly divided into expression profile chip and oligonucleotide chip. Oligonucleotide chip has potential advantages in clinical diagnosis, disease resistance detection, genotyping, etc., such as the study of human mitochondrial 16.6kb genome polymorphism, BRCA I exon 11, CFTR gene, ATM gene, Detection of mutations in HIV-1 reverse transcriptase and protease genes, identification of different species within the genus Mycobacterium, and detection of rifamycin resistance, etc. In view of the advantages of gene chips that require fewer samples to be tested, high ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 韩金祥鲁艳芹
Owner SHANDONG MEDICAL BIO TECH RES CENT
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