Detection genetic chip and detection kit for infectious diarrhea

An infectious diarrhea, gene chip technology, applied in the field of gene chip application, can solve the problems of inapplicability and increase the workload of detection

Inactive Publication Date: 2011-08-03
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the field of pathogenic bacteria detection, many detection technologies based on PCR method have been established at home and abroad, but because PCR technology can only target one or several pathogenic bacteria, and there are many kinds of pathogenic bacteria in water, this requires At

Method used

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  • Detection genetic chip and detection kit for infectious diarrhea
  • Detection genetic chip and detection kit for infectious diarrhea
  • Detection genetic chip and detection kit for infectious diarrhea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1 Probe design and preparation

[0108] 1. Sequence acquisition:

[0109] (1) Bacteria gyrB: Escherichia coli (E.coli) / Shigella (Shigella), Aeromonas hydrophila (Aeromonas hydrophila), Yersinia enterocolitica (Yersinia enterocolitica) were downloaded from the GenBank public database ) of the entire gyrB gene sequence and the entire gyrB gene sequence of its close relatives.

[0110] (2) Acquisition of ITS gene sequences: Download all ITS gene sequences of Salmonella, Vibrio cholerae, Vibrio parahaemolyticus and all ITS genes of their relatives from the GenBank public database sequence.

[0111] (3) Acquisition of dnaJ gene sequences: download all dnaJ gene sequences of Vibrio fluvialis, Vibrio mimicus, Vibrio furnissii and their relatives from the GenBank public database Full dnaJ gene sequence.

[0112] (4) Acquisition of the toxR gene sequence: all toxR gene sequences of Vibriohollisae, Vibrio damsela and their relatives were downloaded from the GenBank ...

Embodiment 2

[0127] Example 2 Primer design and preparation

[0128] 1. Example of primer design:

[0129] (1) Escherichia coli (E.coli) / Shigella (Shigella), hydrophilic Aeromonas (Aeromonashydrophila), enterocolitica (Yersinia enterocolitica) gyrB gene universal amplification primer: the large intestine All the gyrB gene sequences of E.coli / Shigella, Aeromonas hydrophila, and Yersinia enterocolitica were imported into the Glustal X software, and selected A representative sequence was imported into the Primer Primer 5.0 software, and the length was set to 70bp-10bp, the G+C% value was 40%-60%, Hairpin: NONE, Dimer: NONE, False Priming: NONE, Cross Dimer: NONE. And seek out the nucleotide sequence region that is suitable for universal primer design, its characteristic basically meets the following conditions: 1, this constant region should include Escherichia coli (E.coli) / Shigella (Shigella), Aeromonas hydrophilia (Aeromonas hydrophila), gyrB of Yersinia enterocolitica (Yersinia entero...

Embodiment 3

[0146] Example 3 Specific identification of the gene chip

[0147] 1. DNA extraction of target bacteria:

[0148] The collected target bacteria detected by the gene chip and their related strains were cultured overnight for 24 hours; the genomic DNA was extracted with the lysate.

[0149] 2. Amplify the target sequence:

[0150] 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 Tables 3 and 4 below. (Note: PCR buffer, MgCl2, dNTP mixture in the following tables 3, 4-table 5, 6, Taq enzyme are all purchased from Takara company)

[0151] Table 3 PCR reaction mixture AI formula

[0152]

[0153] Note: ① The concentration of each primer in primer mixture I is: P1, P2, P5, P6 is 0.4 μmol L-1; P13, P14 is 0.2 μmol L-1.

[0154] Table 4 PCR reaction mixture BI formula

[0155]

[0156] Note: ①The concentration of each primer in primer mixture...

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Abstract

The invention provides a genetic chip and a detection kit related to major pathogenic microorganisms causing infectious diarrhea of human beings, which are mainly specific to 13 types of bacteria including lapactic bacillus coli/shigella, salmonella, comma bacillus, vibrio parahemolyticus, aeromona hydrophila, plesiomonas, virio hollisae, vibrio fluvialis, vibrio mimicus, Wauter's strains, vibriodamsela and vibrio furnissii. The genetic chip comprises a solid-phase carrier and an oligonucleotide probe fixed on the solid-phase carrier, wherein the oligonucleotide probe comprises DNA (Deoxyribonucleic Acid) fragments selected from genes including a gyrB gene, an ITS gene, a dnaJ gene, a toxR gene and the like with remarkable biological evolution advantages or complementary DNA fragments. By adopting the genetic chip and the detection kit provided by the invention, major pathogenic microorganisms causing infectious diarrhea of human beings can be detected. The genetic chip and the detection kit have the characteristics of easiness and convenience for operation, high flux, high accuracy, high repeatability and the like, and can be applied to clinical detection of inspection departments in hospitals.

Description

technical field [0001] The invention belongs to the technical field of gene chip applications, and relates to a gene chip and a detection kit containing the chip, in particular to a gene chip and a detection kit for the main pathogenic microorganisms causing human infectious diarrhea. Background technique [0002] Infectious diarrhea is one of the infectious diseases with high incidence and widespread prevalence in the world. It poses serious health hazards to humans, especially children, and is an important public health problem in developing countries. According to WHO estimates, tens of millions of people suffer from the disease every day in the world, with 3 to 5 billion cases of diarrhea every year, and 5 to 10 million deaths due to severe diarrhea, with an average of 25,000 deaths per day, especially children. It is prominent that adults in developed countries have diarrhea at least 1 or 2 times a year, and the incidence in developing countries is higher. Diarrhea kil...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor 王磊周光朋刘衍伟曹勃阳窦岩
Owner NANKAI UNIV
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