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Gene chip and kit for detecting common pathogenic bacteria in food

A gene chip and pathogenic bacteria technology, applied in the field of gene chips and kits for detecting seven common pathogenic bacteria in food, can solve the problems of time-consuming, cumbersome, low detection sensitivity, etc., and achieve simple operation, strong repeatability, The effect of high accuracy

Inactive Publication Date: 2012-10-17
TIANJIN BIOCHIP TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011]Traditional food microbiological detection methods mainly include pre-enrichment, selective enrichment, separation, biochemical identification and serotype identification, etc., which generally take 5-7 days to complete , for some atypical suspected positive strains, it takes longer (up to about 10-15 days)
Moreover, different microorganisms have their own different detection procedures, which is a cumbersome, repetitive, time-consuming, laborious and independent technical labor, which makes it difficult to detect food in a timely and high-throughput manner.
At the same time, there is still a key problem that seriously restricts the detection rate of pathogenic microorganisms-the problem of low detection sensitivity, which can easily cause missed detection, so that food containing microbial contamination enters the market, threatening the safety of consumers

Method used

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  • Gene chip and kit for detecting common pathogenic bacteria in food
  • Gene chip and kit for detecting common pathogenic bacteria in food
  • Gene chip and kit for detecting common pathogenic bacteria in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Design and preparation of embodiment 1 probe

[0040] 1. Sequence acquisition:

[0041](1) Acquisition of the invA gene sequence: the entire invA gene sequence of Salmonella was downloaded from the GenBank public database.

[0042] (2) Acquisition of the speB gene sequence: the entire speB gene sequence of Streptococcus pyogenes was downloaded from the GenBank public database.

[0043] (3) Acquisition of the zpx gene sequence: the entire zpx gene sequence of Enterobacter sakazakii was downloaded from the GenBank public database.

[0044] (4) Acquisition of the toxR gene sequence: the entire toxR gene sequence of Vibrio parahaemolyticus was downloaded from the GenBank public database.

[0045] (5) Acquisition of the 16s-23s region gene sequence: the entire 16s-23s region gene sequence of Klebsiella pneumoniae was downloaded from the GenBank public database.

[0046] (6) Acquisition of ipaH gene sequence: the entire ipaH gene sequence of Enterobacter sakazakii was do...

Embodiment 2

[0061] Example 2 Design and preparation of primers

[0062] 1. Sequence acquisition: the same as the sequence of the designed probe.

[0063] 2. Design primers:

[0064] (1) Design of primers for amplifying the specific gene sequence: compare the above-mentioned specific gene sequence downloaded from the GenBank public database with the sequence comparison software Glustal X, find the conserved segment of the gene, and import the conserved segment into the primer design software In Primer Premier 5.0 software, 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, PCR Product Size: 100bp to 1000bp. Primer Length: 20bp±2bp. Search Mode: Automatic. Select T from the output 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 a probe sequence.

[0065] (2) Design of primers for amplifying 16SDN...

Embodiment 3

[0073] Example 3 Gene Chip Preparation——Chip Spotting

[0074] 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 / ml.

[0075] 2. Adding plate: Add the dissolved probe to the corresponding position of the 384-well plate, 10 μl per well.

[0076] 3. Spotting: as figure 1 The shown 57.5mm×25.5mm×1mm (length×width×height) clean aldehydated glass slide is placed on the stage of the chip spotting instrument (Spotarray 72), and the control software of SpotArray is used to run the program, according to figure 2The arrangement shown is spotted on the aldehylated glass slide in the spotting area of ​​4.5mm×4.5mm 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 ​​250μm, the matrix: 12×9...

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Abstract

The invention provides a gene chip and a kit for detecting common pathogenic bacteria in food. The gene chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe comprises DNA sequences which are selected from an nuc gene of staphylococcus aureus, an speB gene of streptococcus pyogenes, an invA gene of salmonella, an ipaHgene of Shigela, a zpx gene of Enterobacter sakazakii, a 16S-23S intergenic region of Klebsiella pneumoniae, a toxR gene of Vibrio parahaemolyticus and 16SDNA of the seven bacteria. The kit comprisesthe gene chip. By utilizing the gene chip and the kit to detect the seven common pathogenic bacteria in food, the invention has the advantages of simple and convenient operation, high accuracy and good 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 seven common pathogenic bacteria in food. Background technique [0002] Foodborne diseases and food contamination are still important factors that endanger public health. In my country, there were 150 cases of food poisoning reported officially in 2000, with 6273 people poisoned and 150 dead. In 2003, there were 379 cases of food poisoning, 12,876 people were poisoned, and 323 people died. Among nearly 10,000 cases of food poisoning every year, except for accidents, most of them are caused by pathogenic microorganisms. For example, in Ningxia in 1999, an outbreak of food poisoning caused by Salmonella contaminated meat occurred, and thousands of people fell ill. Internationally, food poisoning caused by food microorganisms is also a very serious food safety problem. According to incomplete statistics, 75% of the food poisoning...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C40B40/06C12R1/01C12R1/63C12R1/22C12R1/42C12R1/46C12R1/445
Inventor 王磊喻群芳曹勃阳王敏王海波冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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