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Gene chip and kit for detecting diarrheagenic escherichia coli in food and clinical samples

A technology of Escherichia coli and gene chip, which is applied in the field of gene chips and kits for detecting diarrhea-causing E. coli in food and clinical samples, can solve the problems of no correlation of diarrhea-causing E. coli, time-consuming and labor-intensive problems, and achieve accuracy High, easy to operate, repeatable effect

Inactive Publication Date: 2010-06-30
TIANJIN BIOCHIP TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditionally, diarrhea-causing E. coli is mainly detected through biochemical tests and serological tests, but it is time-consuming and laborious. With the deepening of people's research, it is found that diarrhea-causing E. coli is not well correlated with serotypes

Method used

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  • Gene chip and kit for detecting diarrheagenic escherichia coli in food and clinical samples
  • Gene chip and kit for detecting diarrheagenic escherichia coli in food and clinical samples
  • Gene chip and kit for detecting diarrheagenic escherichia coli in food and clinical samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Probe design and preparation

[0037] 1. Sequence acquisition:

[0038] (1) Obtaining the stx1 gene sequence: Download the entire stx1 gene sequence of the diarrheal E. coli from the GenBank public database.

[0039] (2) Obtaining the stx2 gene sequence: Download all the stx2 gene sequences of diarrheal E. coli from the GenBank public database.

[0040] (3) Obtaining the ipaH gene sequence: Download the entire ipaH gene sequence of the diarrheal E. coli from the GenBank public database.

[0041] (4) Obtaining eae gene sequence: downloading all eae gene sequences of diarrheal E. coli from the GenBank public database.

[0042] (5) Obtaining lt gene sequence: Download all lt gene sequences of diarrheal E. coli from the GenBank public database.

[0043] (6) Obtaining the stah gene sequence: Download all the stah gene sequences of the diarrheal E. coli from the GenBank public database.

[0044] (7) Obtaining the stap gene sequence: Download all the stap gene sequences of the...

Embodiment 2

[0064] Example 2 Design and preparation of primers

[0065] 1. Sequence acquisition: same as the sequence of the designed probe.

[0066] 2. Design primers:

[0067] (1) Design of primers for amplification of stx1 gene sequence: The above-mentioned diarrhea-causing E. coli stx1 gene sequence downloaded from the GenBank public database is aligned with the sequence alignment software Glustal X to find the conserved segment of the gene, and the conserved region Import the segment into the primer design software Primer Premier5.0 software, and the corresponding parameter settings are as follows: Search For: PCR Primers, Search types: Both. Search Ranges: Sense Primer 1 to 672, Anti-sense Primer 1 to 672, PCRProduct Size: 100bp to 1000bp.Primer Length:20bp±2bp.Search Mode:Automatic. Select T from the output result m A primer with a value of 50°C±5°C, a length of 17bp±2bp, Hairpin: NONE, Dimer: NONE, False Priming: NONE, Cross Dimer: NONE, and including the probe sequence.

[0068] (2)...

Embodiment 3

[0081] Example 3 Gene chip preparation-chip spotting

[0082] 1. Dissolving the probes: the probes synthesized in Example 1 were respectively dissolved in 50% DMSO solution, and diluted to make the final concentration of the probe reach 1 μg / μl.

[0083] 2. Add plate: add the dissolved probe to the corresponding position of the 384-well plate, 10μl per well.

[0084] 3. Pointing: Will be as figure 1 The shown 57.5mm×25.5mm×1mm (L×W×H) clean aldehyde-based glass slide (CEL Associates, Inc.) is placed on the stage of the chip spotter (Spotarray 72), and SpotArray is used Control software (Tele chem smp3 stealty pin), run the program, press figure 2 The arrangement shown is dotted in a 4.5mm×4.5mm spot area on the aldehyde-based glass slide, forming a medium and low density DNA micro-matrix. The six dot matrix areas on the glass slide have the same array arrangement. The size of the dot matrix area is 3mm×2.25mm, the dot pitch in the dot matrix is ​​250μm, the matrix: 12×9, 12×250μ...

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Abstract

The invention provides a gene chip and a kit for detecting diarrheagenic escherichia coli in food and clinical samples. The gene chip comprises a solid-phase carrier and an oligonucleotide probe fixed on the solid-phase carrier, wherein the oligonucleotide probe contains DNA fragments and the complementary DNA or RNA sequence selected from eae genes of enteropathogenic escherichia coli (EPEC), lt, STap and STah genes of enterotoxigenic escherichia coli (ETEC), stx1 and stx2 of enterohaemorrhagic escherichia coli (EHEC), ipaH genes of enteroinvasive escherichia coli (EIEC), escherichia coli O157wzy genes and escherichia coli 16s genes. The invention further provides the kit containing the gene chip. The utilization of the gene chip and the kit for detecting the diarrheagenic escherichia coli has the advantages of simple operation, high accuracy and strong repeatability.

Description

Technical field [0001] The invention relates to a gene chip and a detection kit, in particular to a gene chip and a kit for detecting diarrheal E. coli in food and clinical samples. Background technique [0002] According to statistics from the World Health Organization, apart from China, 1 billion people worldwide suffer from diarrhea each year, of which 500 million occur in the third world, causing 5 million infant deaths each year; 836 million people suffer from diarrhea each year in China, of whom 5 years old The following children have 300 million visits. At present, there are dozens of pathogens known to cause diarrhea, and bacterial and viral diarrhea are the majority of reports in China. Although the main pathogens that cause diarrhea in areas with different natural environments and different production and lifestyles are different, statistics show that diarrheal E. coli ranks second and is the main diarrheal pathogen. [0003] Five types of enterodiarrheal E. coli have b...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10
Inventor 王磊曾静孙亚民曹勃阳王敏喻群芳
Owner TIANJIN BIOCHIP TECH CO LTD
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