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Gene chip of main pathogenic microorganism in drinking water and testing kit

A gene chip and drinking water technology, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, resistance to vector-borne diseases, etc., can solve problems such as complex operations

Inactive Publication Date: 2010-06-23
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method also involves the multiple PCR problem of more than 6 pairs of primers, and the operation is complicated

Method used

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  • Gene chip of main pathogenic microorganism in drinking water and testing kit
  • Gene chip of main pathogenic microorganism in drinking water and testing kit
  • Gene chip of main pathogenic microorganism in drinking water and testing kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Probe design and preparation

[0088] 1. Sequence acquisition:

[0089] (1) Bacteria gyrB: Escherichia coli (E.coli) / Shigella (Shigella), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Legionella pneumophila (Legionella pneumophila), small intestine colon All gyrB gene sequences of Yersinia enterocolitica and all gyrB gene sequences of its close relatives.

[0090] (2) Acquisition of ITS gene sequences: download Salmonella, Vibrio cholerae, Vibrioparahaemolyticus, Staphylococcus aureus, Klebsiella pneumoniae from the GenBank public database All ITS gene sequences of Klebsiella pneumoniae and all ITS gene sequences of its close relatives.

[0091] (3) Acquisition of 16s rRNA gene sequence: the entire 16s rRNA gene sequence of Enterococcus faecalis and all 16s rRNA gene sequences of its close relatives were downloaded from the GenBank public database.

[0092] (4) Acquisition of the gyrB gene sequence of Leptospira interrogans: the entire gyrB gene seque...

Embodiment 2

[0105] Example 2 Primer design and preparation

[0106] 1. Example of primer design:

[0107] (1) Escherichia coli (E.coli) / Shigella (Shigella), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Legionella pneumophila (Legionella pneumophila), Yersinia enterocolitica (Yersinia enterocolitica) ) gyrB gene universal amplification primers: Escherichia coli (E.coli) / Shigella (Shigella), Pseudomonas aeruginosa (Pseudomonasaeruginosa), Legionella pneumophila (Legionella pneumophila), Yersinia enterocolitica All the gyrB gene sequences of Yersinia enterocolitica were imported into the Glustal X software, and a representative sequence was selected and imported into the Primer Primer 5.0 software. : NONE, Dimer: NONE, FalsePriming: NONE, Cross Dimer: NONE. And seek out the nucleotide sequence region that is suitable for general primer design, its characteristic basically meets the following conditions: 1, this constant region should comprise Escherichia coli (E.coli) / Shigella (Shig...

Embodiment 3

[0120] Example 3 Rapid detection of main pathogenic microorganisms in drinking water by gene chip and preparation of kit

[0121] 1. Sample handling:

[0122] (1) Proliferate the collected environmental samples on a universal medium for 12 hours;

[0123] (2) Centrifuge at 15000g for 10 minutes

[0124] (3) Discard the supernatant, add 100 μl of lysate, mix well, and bathe in water at 100°C for 10 minutes;

[0125] (4) The lysate obtained in the previous step was centrifuged at 15000g for 5 minutes;

[0126] (5) Collect the supernatant, which contains genomic DNA and can be used for detection or stored at -20°C.

[0127] Attachment: lysate formula:

[0128] 50mmolL-1NaOH

[0129] 10mmolL-1Tris-HCl (pH 8.0)

[0130] 0.5% Tween-20

[0131] 0.5% NP-40

[0132] 0.5m molL-1EDTA (pH 8.0)

[0133] 5% chelex-100

[0134] 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...

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Abstract

The invention provides a gene chip of main pathogenic microorganism in drinking water and a testing kit, which mainly aims at 11 kinds of bacteria of colibacillus / Shigella, salmonella, vibrio cholera, vibrio parahaemolyticus, staphylococcus aureus, enterococcus faecails, pseudomonas aeruginosa, legionella pneumophilia, pneumobacillus, yersinia enterocolitica and the like, and L.interrogans. The gene chip comprises a solid phase carrier and a oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe contains gyrB gene with tremendous evolutionary advantage, ITS gene and DNA segment selected from 16srRNA gene or complementary DNA segment. The gene chip and the testing kit of the invention can test the main pathogenic microorganism in drinking water, and has the characteristics of simple operation, high throughput, high accuracy, strong repeatability and the like, and can be used for clinical test for the water quality monitoring department.

Description

technical field [0001] The invention relates to a gene chip and a detection kit containing the chip, in particular to a gene chip and a detection kit for main pathogenic microorganisms in drinking water. Background technique [0002] Water is a necessary condition for the existence of life. It enables us to multiply and thrive. Human life, production and entertainment cannot do without water. It is also a place and carrier for many pathogenic microorganisms to breed and spread. Once these pathogenic microorganisms enter the human body, they may cause illness or even death, seriously threatening human health. With the development of society and the improvement of living standards, people are more and more concerned about their own health issues, and the safety of various water bodies (including drinking water, rivers and lakes, swimming pools, etc.) has increasingly become a focus of attention. Therefore, in order to protect people's health, it is very necessary and urgent t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04
CPCY02A50/30
Inventor 曹勃阳周光朋王磊闻少平朱之燕
Owner NANKAI UNIV
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