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Pathogenic microorganism DNA detecting chip and preparation method

A technology for pathogenic microorganisms and detection chips, which can be used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., and can solve problems such as hindering food import and export.

Inactive Publication Date: 2010-05-26
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This greatly hinders the import and export of food

Method used

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  • Pathogenic microorganism DNA detecting chip and preparation method
  • Pathogenic microorganism DNA detecting chip and preparation method
  • Pathogenic microorganism DNA detecting chip and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: Preparation of pathogenic microorganism detection chip

[0061] 1. Extraction of genomic DNA of pathogenic microorganisms: the following pathogenic microorganisms are extracted routinely: Vibrio cholerae, pathogenic Escherichia coli, Campylobacter jejuni, Yersinia enterocolitica, Vibrio parahaemolyticus, Salmonella, Shigella, Listeria DNA. (See A Laboratory Guide to Molecular Cloning (Third Edition), Cold Spring Harbor Press)

[0062] 2. Screening of primers and preparation of detection probes

[0063] Using the above-mentioned pathogenic microorganism genomic DNA as a template, use the primers corresponding to each strain (see Table 1, synthesized by Beijing Saibaisheng Gene Technology Co., Ltd., Shanghai Sangon Bioengineering Co., Ltd. and Beijing Aoke Biotechnology Co., Ltd.) The strain was amplified by PCR (TaqDNA polymerase, purchased from Beijing Broadtech Biogene Technology Co., Ltd.), and the obtained product was identified by agarose gel electro...

Embodiment 2

[0087] Example 2: Marking of the target gene in the sample to be tested

[0088] (1) Prepare the sample to be tested according to the method described in Example 1;

[0089] (2) The samples are labeled and purified by multiplex PCR amplification and then hybridized with the chip. Multiplex PCR fluorescein-labeled sample PCR reaction system 25 μl, the primers are mixed primers, combination 1, combination 2, and combination 3, the concentration is 1.6 μM, the samples to be tested are labeled with three sets of primers, and then the labeled products are mixed and purified.

Embodiment 3

[0090] Embodiment 3: the detection of sample to be tested

[0091] 1. Hybridization detection chip based on PCR product immobilization method

[0092] Steps:

[0093] (1) Place the slide in the hybridization chamber.

[0094] (2) Heat and denature the microcentrifuge tube containing the labeled probe: cook in boiling water at 100° C. for 3 minutes.

[0095] (3) Add 100 μl of high-efficiency hybridization solution (Broadtech Co., Ltd.) into the tube, mix well by pipetting, and pour into the hybridization chamber. Hybridization was performed at 42°C for 18 hours.

[0096] (4) After 18 hours, the slides were taken out, and put into membrane washing solution I (2×SSC, 0.1% SDS) to wash for 2 minutes.

[0097] (5) Wash the membrane with II solution (0.1×SSC, 0.1% SDS) for 2 minutes.

[0098] (6) Wash with ultrapure water for 2 minutes, dry and wait for scanning.

[0099] 2. Chip cleaning

[0100] (1) Wash in membrane washing solution I (2×SSC, 0.1% SDS) for 2 minutes.

[01...

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Abstract

The invention relates to a pathogenic microorganism DNA detection chip and the chip comprises a carrier and a nucleic acid probe on the carrier. The nucleic acid probe is provided with a target detection probe used for detecting target gene of pathogenic microorganism. The pathogenic microorganism comprises but not limits to vibrio cholerae, pathogenic escherichia coli, campylobacter jejuni, Yersinia enterocolitica, parahemolytic vibrio, salmonella, and shigella and Listeria monocytogenes. The invention also relates to a preparation method of the pathogenic microorganism DNA detection chip andprovides applications of the pathogenic microorganism DNA detection chip in detection of pathogenic microorganism. Also a pathogenic microorganism DNA detection chip kit is provided.

Description

[0001] The invention is based on the following funded projects: [0002] 1. Topic 1: National Natural Science Foundation of China Grant No.: 30170052 "Molecular Biology of Campylobacter jejuni Associated with Guillain-Barré Syndrome" 2002.01-2004.12: [0003] 2. Topic 2: Social Welfare Project of the Ministry of Science and Technology: "Establishment of National Rapid Inspection Method System for Imported and Exported Foods" 2003; Project Number: 2002DIA50036; [0004] 3. Topic 3: Social welfare project of the Ministry of Science and Technology: "Establishment of a rapid detection system for pathogens and genetically modified labels in food" 2005; project number: 2004DIB2J065. technical field [0005] The invention relates to the field of biotechnology, in particular to a pathogenic microorganism DNA detection chip and its preparation method and application. Background technique [0006] Infectious diseases caused by microorganisms are still the main diseases threatening h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCY02A50/30
Inventor 张建中尤元海曾浔姜海肖迪闫笑梅尹焱
Owner ICDC CHINA CDC
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