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Gene chip for detecting important pathogenic bacteria in aquatic product and kit thereof

A technology of gene chips and aquatic products, applied in the field of gene chips and kits for detecting important pathogenic bacteria in aquatic products, can solve the problems of complex operation, long inspection cycle, heavy manual labor, etc., achieve strong repeatability, The effect of high accuracy and easy operation

Inactive Publication Date: 2012-01-11
TIANJIN BIOCHIP TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection and identification methods of pathogenic bacteria in aquatic products in my country still remain at the conventional microbiological inspection level, usually based on isolation culture, biochemical tests and serological tests, which have complex operations, heavy manual labor, and long inspection cycles (6-7 Days), low specificity and other shortcomings, to a considerable extent, limit the rapid detection and identification of aquatic products of foodborne diseases, and when judging whether it is positive or not, only rely on the naked eye of the experimenter, there is no reliable enough basis, reduce the accuracy of the experimental results, and cannot meet the requirements of short storage time and rapid detection of fresh aquatic products
Some simple molecular biology methods such as PCR and ELISA have high false positive results, and the results obtained by using PCR and ELISA cannot be used as the final detection basis

Method used

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  • Gene chip for detecting important pathogenic bacteria in aquatic product and kit thereof
  • Gene chip for detecting important pathogenic bacteria in aquatic product and kit thereof
  • Gene chip for detecting important pathogenic bacteria in aquatic product and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Design and preparation of embodiment 1 probe

[0037] 1. Sequence acquisition:

[0038] (1) Acquisition of bacterial 16S rDNA sequences: twelve species of bacteria (Proteus mirabilis, Proteus vulgaris, Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus) were downloaded from the GenBank public database. coccus, Streptococcus pyogenes, Vibrio vulnificus, Bacillus cereus group, Shigella, Yersinia enterocolitica) all 16S rDNA sequences.

[0039] (2) Acquisition of 16S-23S rDNA interregional sequences: all 16S-23S rDNA interregional sequences of Proteus mirabilis, Proteus vulgaris and their relatives were downloaded from the GenBank public database.

[0040] (3) Acquisition of ipaH gene sequences: all ipaH gene sequences of the four species of Shigella were downloaded from the GenBank public database.

[0041] (4) Acquisition of the invA gene sequence: the entire invA gene sequence of Salmonella was downloaded from the Gen...

Embodiment 2

[0059] Design and preparation of embodiment 2 primers

[0060] 1. Sequence acquisition: the same as the sequence of the designed probe.

[0061] 2. Design primers:

[0062] (1) Design of primers for amplifying the interregional sequence: After comparing the 16S rDNA of twelve species of bacteria downloaded from the public database NCBI with the sequence alignment software Glustal X, a 16S rDNA conservative sequence close to the interregional region was selected as the upstream Primers, lengths conforming to T m Value 50°C±5°C, length 17bp±2bp, Hairpin: NONE, Dimer: NONE, False Priming: NONE, CrossDimer: NONE, and contains bacterial universal probe sequence.

[0063] (2) Design of primers for amplifying specific gene sequences: compare the above-mentioned specific gene sequences of ten species of bacteria except Proteus mirabilis and Proteus vulgaris downloaded from the GenBank public database with the sequence comparison software Glustal X, Find the conserved segment of t...

Embodiment 3

[0069] Example 3 Gene Chip Preparation——Chip Spotting

[0070] 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.

[0071] 2. Adding plate: Add the dissolved probe to the corresponding position of the 384-well plate, 10 μl per well.

[0072] 3. Spotting: as figure 1 The shown 57.5mm × 25.5mm × 1mm (length × width × height) clean aldehyde slide (CapitalBio Corp., China) was placed on the stage of the chip spotter (Spotarray 72), using SpotArray control software (Telechem 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×2mm, the dot pitch in the dot matrix is ​​250μ...

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Abstract

The invention relates to a gene chip for detecting important pathogenic bacteria in an aquatic product and a kit thereof. The gene chip comprises a solid phase carrier and an oligonucleotide probe; the oligonucleotide probe comprises one or more of the following nucleotide sequences: (1) DNA (Deoxyribonucleic Acid) sequences selected from proteus mirabilis 1, proteus vulgaris, salmonella, vibrio parahaemolyticus, vibrio cholerae, Listeria monocytogenes, staphylococcus aureus, streptococcus pyogenes, vibrio vulnificus, bacillus cereus cluster, Shigella and pathogenic Y. enterocolitica ail gene; (2) complementary DNA sequences of the DNA sequences selected in the (1); (3) complementary RNA (Ribonucleic Acid) sequences of the DNA sequences in the (1) or (2). The kit comprises the gene chip. The gene chip and the kit can be used for detecting the important pathogenic bacteria in an aquatic product, are convenient to operate, high in precision and strong in repeatability.

Description

technical field [0001] The invention relates to a gene chip and a kit for detection, in particular to a gene chip and a kit for detecting important pathogenic bacteria in aquatic products. Background technique [0002] Food quality and food safety are related to human health, so they are highly valued by countries all over the world. Foodborne illnesses caused by microorganisms are a major problem in food safety. Foodborne disease (food poisoning) caused by bacterial contamination is the most prominent problem in my country's food safety. According to statistics from the Ministry of Health, in 2000 there were 150 major food poisoning reports, 6,237 people were poisoned, and 135 people died; in 2001, there were 185 major food poisoning incidents, 15,715 people were poisoned, and 116 people died. Among the food poisoning incidents reported all over the country in 2005, microbial food poisoning accounted for the largest number, accounting for 43.0% of the total. The data ana...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 王磊李荣荣张恋心曹勃阳冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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