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Gene chip and test kit for detecting important pathogenic bacteria in aquatic products

A gene chip and pathogenic bacteria technology, applied in the field of gene chips and kits for detecting important pathogenic bacteria in aquatic products, can solve problems such as identification difficulties, and achieve the effects of simple operation, strong repeatability and high accuracy

Inactive Publication Date: 2012-02-29
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacterial microorganisms can be identified to a species or genus using 16S rRNA and 23S rRNA, but it is very difficult to identify bacterial species or genus with close relatives (BodrossyL, Sessitsch A. 2004. Oligonucleotide microarrays in microbial diagnostics. Current Opinion in Microbiology. 7:245-25)

Method used

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  • Gene chip and test kit for detecting important pathogenic bacteria in aquatic products
  • Gene chip and test kit for detecting important pathogenic bacteria in aquatic products
  • Gene chip and test kit for detecting important pathogenic bacteria in aquatic products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Design and preparation of probes

[0035] 1. Sequence acquisition:

[0036] (1) Acquisition of bacterial 16S rDNA sequences: all 16S rDNA sequences of ten species of bacteria were downloaded from the GenBank public database.

[0037] (2) Acquisition of 16S-23S rDNA interval sequence: download from the GenBank public database to obtain Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, and their All 16S-23S rDNA interregion sequences of closely related bacteria.

[0038] In order to meet the needs of intra-species conservation and inter-species specificity in target sequence analysis, 48 ​​strains of Proteus (including all 4 species of this genus) were selected for the genus Proteus with few ITS resources: 22 strains of Proteus mirabilis, 14 strains of Proteus common strains, 10 strains of Proteobacterium panethii, and 2 strains of Proteus myxogenes) were sequenced in the interregion,...

Embodiment 2

[0051] Example 2 Design and preparation of primers

[0052] 1. Sequence acquisition: the same as the sequence of the designed probe.

[0053] 2. Design primers:

[0054] (1) Design of primers for amplifying the interregional sequence: After comparing the 16S rDNA of ten species of bacteria downloaded from the public database NCBI with the sequence alignment software Glustal X, a 16S rDNA conserved sequence close to the interregional region was selected as the upstream primer , the length conforms 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.

[0055] (2) Design of primers for amplifying the ipaH gene sequence: the above-mentioned ipaH gene sequences of the four species of Shigella downloaded from the GenBank public database were compared with the sequence alignment software Glustal X to find the conserved segment of the gene, The conserved segment was imported int...

Embodiment 3

[0061] Example 3 Gene Chip Preparation——Chip Spotting

[0062] 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.

[0063] 2. Adding plate: Add the dissolved probe to the corresponding position of the 384-well plate, 10 μl per well.

[0064] 3. Spotting: as figure 1 The shown 57.5mm × 25.5mm × 1mm (length × width × height) of the clean formaldehyde slide (CapitalBio Corp., China) was placed on the stage of the chip spotting instrument (Spotarray72), using SpotArray's Control software (Telechem smp3 stealty pin), run the program, press figure 2The 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...

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Abstract

The invention relates to a gene chip and a test kit for detecting important pathogenic bacteria in aquatic products, wherein the gene chip comprises a solid matrix and an oligonucleotide probe composed of one or more serials selected from the following serials: (1) DNA serials selected from the spacers of 16S-23rS DNA of salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, staphylococcus aureus, Suppurative streptococcus, Proteus mirabilis, Proteus Vulgaris and Paneth Proteus, and ipaH Virulence gene of Shigella; (2) complement DNA serials of the DNA serials selected from (1); (3) complement RNA serials of the DNA serials selected from (1) or (2). The chip and the test kit comprise simple and convenient operation, high veracity and strong repeatability in detecting the important pathogenic bacteria in aquatic products.

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 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. Th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04
CPCC12Q1/6837G01N2035/00158C12Q1/689C12Q2600/166Y02A50/30
Inventor 王磊刘蕾曹勃阳王敏
Owner TIANJIN BIOCHIP TECH CO LTD
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