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Gene probe for simultaneously detecting five food-borne pathogenic bacteria through liquid chip and detection method

A food-borne pathogenic bacteria, liquid-phase chip technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial assay/inspection, etc., to achieve the effects of low cost, strong primer specificity, and strong stability

Inactive Publication Date: 2015-12-30
GUANGZHOU CENT FOR DISEASE CONTROL & PREVENTION (GUANGZHOU HYGIENE INSPECTION CENT GUANGZHOU CENT FOR FOOD SAFETY RISK SURVEILLANCE & ASSESSMENT INST OF PUBLIC HEALTH OF GUANGZHOU MEDICAL UNIV)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] One of the purposes of the present invention is to solve the problem of how to simultaneously detect five kinds of foodborne pathogenic bacteria using a liquid phase chip. The present invention provides a primer and detection method for simultaneously detecting five kinds of foodborne pathogenic bacteria using a liquid phase chip Methods: Using the LuminexxMAP liquid chip system and strain library resources, a rapid detection of five common foodborne pathogens (including Salmonella, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus) was established. Bacteria), providing a scientific basis for the further application of liquid chip technology in the rapid and effective prevention and control of foodborne diseases

Method used

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  • Gene probe for simultaneously detecting five food-borne pathogenic bacteria through liquid chip and detection method
  • Gene probe for simultaneously detecting five food-borne pathogenic bacteria through liquid chip and detection method
  • Gene probe for simultaneously detecting five food-borne pathogenic bacteria through liquid chip and detection method

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Embodiment 1

[0055] A gene probe for simultaneous detection of five food-borne pathogenic bacteria by using a liquid chip, including the following gene probes for detecting five food-borne pathogens:

[0056] Probes for the detection of the invasion protein invA gene of Salmonella:

[0057] The nucleotide sequence of its probe is NH 2 -5'TTTTTTTTTTTTTACCTATCTGGTTGATTTCCTG-3';

[0058] Probes for the detection of the Hly gene of Listeria monocytogenes:

[0059] The nucleotide sequence of its probe is NH 2 -5'TTTTTTTTTTTTTCGCCTGCAAGTCCTAAGA-3';

[0060] Probes for the detection of the sa442 gene of Staphylococcus aureus:

[0061] The nucleotide sequence of its probe is NH 2 -5'TTTTTTTTTTTTTTGCATCGGAAACATTGT-3';

[0062] Probes for detecting the hlyA gene of Vibrio cholerae:

[0063] The nucleotide sequence of its probe is NH 2 -5'TTTTTTTTTTTTATGCGATTGCCCAAGA-3';

[0064] Probes for detecting the toxR gene of Vibrio parahaemolyticus:

[0065] The nucleotide sequence of its probe is ...

Embodiment 2

[0085] Step 4: Coupling the gene probes of five kinds of food-borne pathogenic bacteria with the microspheres to obtain the microsphere probes and verification, the verification method and results are shown in Example 2;

[0086] The coupling of probes and microspheres is carried out according to the operation steps recommended by Luminex:

[0087] ①. Take a fresh EDC powder frozen at -20°C and packed at room temperature.

[0088] ②. The modified -NH 2 The oligonucleotides were diluted with sterilized deionized water and resuspended to 1 mmol / L, which was the storage concentration.

[0089] ③. Take the concentration as 5.0×10 6 pcs / mL R&D microspheres or color-coded microspheres, shake to mix, and sonicate for 20s to resuspend the microspheres.

[0090] ④. Pipette 200uL microspheres into a brown Eppendorf tube, centrifuge the microspheres at 8000g for 2min, discard the supernatant.

[0091] ⑤. Add 50 ul of MES (pH 4.5) with a concentration of 0.1 mol / L, shake to mix, and s...

Embodiment 3

[0120] Example 3 Optimization of liquid phase chip hybridization temperature

[0121] The optimal conditions for hybridization of oligonucleotide probes are to ensure the specificity of hybridization and to form stable hybrids at an appropriate rate. The longer the probe sequence, the easier it is to form a hybrid, but it lacks specificity and the hybridization rate slows down. Short probes have higher specificity but suffer from unstable hybrid defects. Temperature has a significant impact on the hybridization effect. Selecting the most appropriate hybridization temperature can enhance specific hybridization and reduce the fluorescence intensity of non-specific hybridization. In this study, five different probes were used as the research objects to optimize the hybridization temperature.

[0122] Configure the microsphere solution: dilute the microsphere probe with the microsphere diluent, and prepare 5 times according to the test volume: take 100 μL of the microsphere dilu...

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Abstract

The invention relates to the technical field of security detection of agricultural and sideline products, in particular to a primer for simultaneously detecting five food-borne pathogenic bacteria through a liquid chip and a detection method. The detection method comprises steps as follows: a specific primer and a gene probe for five foodborne pathogenic bacteria are designed; DNA templates of the detected five foodborne pathogenic bacteria are prepared; PCR (polymerase chain reaction) amplification is performed on the five foodborne pathogenic bacteria; coupling is performed on the gene probe for the five foodborne pathogenic bacteria and microspheres, and a microsphere probe is obtained and verified; an obtained PCR amplification product and the microsphere probe have a hybridization reaction; a fluorescence signal of a hybridized product is detected, and quantitative analysis is performed on the five foodborne pathogenic bacteria. A method capable of simultaneously detecting five common foodborne pathogenic bacteria is successfully established, the variable coefficient is within 2%, the specificity is 100%, the sensitivity can reach 100 CFU / mL, and the method is equivalent to a fluorescent PCR technology and can be applied to high-throughput rapid detection of clinic and environment specimens of foodborne diseases.

Description

technical field [0001] The invention relates to the technical field of safety detection of agricultural and sideline products, in particular to a primer and a detection method for simultaneously detecting five kinds of food-borne pathogenic bacteria by using a liquid phase chip. Background technique [0002] The World Health Organization (WHO) defines foodborne disease as infection or poisoning caused by various pathogenic factors that enter the human body through food. According to WHO estimates, billions of people suffer from foodborne diseases every year worldwide, resulting in the death of about 3 million children under the age of 5. In developed countries, one-third of the population is infected with foodborne diseases every year. In developing countries, it is estimated that there are 21.7 billion cases of diarrhea and related diseases every year, resulting in the death of 2.4 million children under the age of 5, causing huge health hazards and economic burdens. Nine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04C12N15/11C12R1/42C12R1/445C12R1/63C12R1/01
CPCC12Q1/6806C12Q1/6834C12Q1/689C12Q2600/16C12Q2563/107C12Q2563/149C12Q2565/519C12Q2531/113C12Q2537/143Y02A50/30
Inventor 吴新伟肖性龙伍业健陶霞杨智聪
Owner GUANGZHOU CENT FOR DISEASE CONTROL & PREVENTION (GUANGZHOU HYGIENE INSPECTION CENT GUANGZHOU CENT FOR FOOD SAFETY RISK SURVEILLANCE & ASSESSMENT INST OF PUBLIC HEALTH OF GUANGZHOU MEDICAL UNIV)
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