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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 time-consuming and labor-intensive, no correlation of diarrhea-causing E. coli, and achieve easy operation , High accuracy and repeatable effect

Inactive Publication Date: 2012-06-27
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) Acquisition of stx1 gene sequence: the entire stx1 gene sequence of diarrhea-causing Escherichia coli was downloaded from the GenBank public database.

[0039] (2) Acquisition of stx2 gene sequence: the entire stx2 gene sequence of diarrhea-causing Escherichia coli was downloaded from the GenBank public database.

[0040] (3) Acquisition of ipaH gene sequence: the entire ipaH gene sequence of diarrhea-causing Escherichia coli was downloaded from the GenBank public database.

[0041] (4) Acquisition of eae gene sequences: all eae gene sequences of diarrhea-causing Escherichia coli were downloaded from the GenBank public database.

[0042] (5) Acquisition of lt gene sequences: all lt gene sequences of diarrhea-causing Escherichia coli were downloaded from the GenBank public database.

[0043] (6) Acquisition of stah gene sequences: all stah gene sequences of diarrhea-causing Escheri...

Embodiment 2

[0064] Example 2 Primer design and preparation

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

[0066] 2. Design primers:

[0067] (1) Design of primers for amplifying the stx1 gene sequence: compare the above-mentioned diarrheagenic Escherichia coli stx1 gene sequence downloaded from the GenBank public database with the sequence comparison software Glustal X, find the conserved segment of the gene, and use the conserved region The segment is imported into the primer design software Primer Premier5.0 software, and the corresponding parameters are set 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 mA 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 a probe sequence.

[0068] (2) De...

Embodiment 3

[0082] Example 3 Gene Chip Preparation - Chip Spotting

[0083] 1. Dissolving probes: the probes synthesized in Example 1 were respectively dissolved in 50% DMSO solution, and diluted so that the final concentration of the probes reached 1 μg / μl.

[0084] 2. Adding plate: Add the dissolved probe to the corresponding position of the 384-well plate, 10 μl per well.

[0085] 3. Spotting: as figure 1The shown 57.5mm × 25.5mm × 1mm (length × width × height) clean aldehyde slide (CEL Associates, Inc.) was placed on the stage of the chip spotter (Spotarray 72), using SpotArray Control software (Tele chem smp3 stealty pin), run the program, press figure 2 The arrangement shown is spotted on the aldehydeized glass slide in the spotting area of ​​4.5 mm×4.5 mm to form a medium-low density DNA micro-array, and the array arrangement rules in the six dot matrix areas on the glass slide are the same. The size of the dot matrix area is 3mm×2.25mm, the dot pitch in the dot matrix is ​​2...

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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 test kit for detection, in particular to a gene chip and a test kit for detecting diarrhea-causing Escherichia coli in food and clinical samples. Background technique [0002] According to the statistics of the World Health Organization, except China, there are 1 billion people suffering from diarrhea every year in the world, of which 500 million people occur in the third world, resulting in the death of 5 million children every year; 836 million people suffer from diarrhea every year in China, of which 5 years old The following children have 300 million person-times. At present, there are dozens of pathogens known to cause diarrhea, and bacterial and viral diarrhea account for the majority of domestic reports in China. Although the main pathogens causing diarrhea are different in areas with different natural environments and different production and lifestyles, statistics show that diarrhea-causing Escherich...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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