Preparation method for biological chip for detecting food-borne pathogenic bacteria

A technology of food-borne pathogenic bacteria and biochips, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, organic compound libraries, etc., can solve heavy preparation and finishing work, long detection cycle, lack of specificity, etc. problems, avoiding the interaction between primers and products, ensuring specificity, and reducing costs

Inactive Publication Date: 2017-07-28
XUZHOU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the improvement of inspection requirements and the expansion of inspection scale, the shortcomings of traditional methods have become increasingly prominent, such as long inspection cycle, heavy preparation and finishing work, insufficient specificity, low sensitivity, and the need for professional operators, etc.

Method used

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  • Preparation method for biological chip for detecting food-borne pathogenic bacteria
  • Preparation method for biological chip for detecting food-borne pathogenic bacteria
  • Preparation method for biological chip for detecting food-borne pathogenic bacteria

Examples

Experimental program
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Effect test

preparation example Construction

[0028] A method for preparing a chip for detecting food-borne pathogenic microorganisms, the specific steps are as follows:

[0029] (1): Soak the silanized glass slide in 5% glutaraldehyde (50% aqueous solution, Amresco) phosphate buffer solution (0.1M PH7.4) for 2 hours, wash with acetone, ethanol, deionized distilled water Thoroughly wash each twice, blow dry with nitrogen, and store at 4°C for later use;

[0030] (2): Design and synthesis of probes:

[0031] TP: 5′-GCCTTTACAGATAGCATGCCATACAGTCATTTCACGC-3′

[0032] CP: 5′-NH 2 -TTTTTTTTGCGTGAAATGACTGTATGG-3′

[0033] DP: 5′-CATGCTATCTGTAAAGGCTTTCGTTTCACGACGAAATAACGGGATACAGGAC-3′

[0034] RCP: 5′-CGTTATTTCGTCTATTCTACTGTATTCTGTATGTCTCCGTATCGCTGTCACCTGTGTATCTTTGATTCGTCAGTCCTGTATCC-3′

[0035] PC:5′-NH 2 -TTTTTTTTTTGCATATGAGCGTCTCGACTTT-biotin-3'

[0036] NC: 5′-NH 2 -TTTTTTTTTTTCGTTACTAGTACATGCTTCGGTTT-3′

[0037] (3): CP, PC and NC were prepared as a 100 μM stock solution using TE buffer and stored at -20°C. Before ...

Embodiment 2

[0050] Embodiment 2: Verify the feasibility and specificity of the microarray chip detection method:

[0051] (1) Spot PC, NC and CP onto the aldehyde-based substrate using a spotting instrument, with a probe concentration of 2 μM.

[0052] (2) The probe fixing process is the same as (1)-(4) in Example 1.

[0053] (3)(3) Add TP, DP (final concentration 0.05μM) and RCP (5μL) into the hybridization solution in the EP tube, heat at 95°C, denature for 5min, and quickly transfer 25μL of the hybridization reaction system in the EP tube to the aldehyde In the 25 μL Gene Frame of the substrate, cover the cover glass, place it in a hybridization oven to start the hybridization reaction, and hybridize at 50°C for 4 hours.

[0054] (4) connection, RCA, washing and scanning, the steps are the same as (6)-(12) of Example 1.

[0055] Taking TP as the detection object, TP was directly added to the hybridization reaction system for microarray chip rolling circle amplification detection, and...

Embodiment 3

[0056] Embodiment 3: Detection effect of Staphylococcus aureus

[0057] Staphylococcus aureus (CMCC26003) was cultured, and gDNA of Staphylococcus aureus was extracted.

[0058] The sampling steps are the same as (1)-(12) of Example 1.

[0059] Cultivate Staphylococcus aureus (CMCC26003), extract its gDNA, prepare 2μM CP, PC and NC, and array, and perform sRCA reaction. The detection results are as follows: Figure 4 As shown, both PC and CP have fluorescence signals, and NC has no fluorescence signals, indicating that the method of the present invention can specifically detect Staphylococcus aureus gDNA, indicating that the detection method has high specificity.

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Abstract

The invention discloses a preparation method for a biological chip for detecting food-borne pathogenic bacteria and belongs to the technical field of food-borne pathogenic microorganism detection. According to the invention, the unique protein gene sequence of food-borne pathogenic bacteria is utilized to design the specific CP, DP and RCP thereof. CP and DP both contain the sequences complementary with the specific sequence of to-be-detected bacterium genome DNA; the RCP contains no to-be-detected bacterium genome DNA sequences; the CP is fixed on a formyl-modified chip surface in a sample application mode, a capturing probe micro-array is prepared, on-chip CP and DP are annealed and connected with the to-be-detected bacterium genome DNA, streptavidin is added and is combined with a solid-phase RCA amplification product and elution is performed under a certain condition; a near infrared fluorescence imager is used for scanning the chip and is used for detecting if CP and DP are connected with each other and if the rolling circle amplification and target detection can be realized, so that the target food-borne pathogenic bacteria can be specifically detected. The method can be used for detecting various food-borne pathogenic microorganisms and is suitable for high-flux, quick and accurate detection for various food-borne pathogenic microorganisms in food, breeding, ports, and the like.

Description

technical field [0001] The invention relates to a detection technology for food-borne pathogenic microorganisms, in particular to a method for preparing a biochip for detecting food-borne pathogenic microorganisms. Background technique [0002] Common foodborne pathogens mainly include: Salmonella, Vibrio parahaemolyticus, Staphylococcus aureus, Clostridium botulinum, E. Caterpillars, Aspergillus flavus, etc. The types of food-borne pathogens are still increasing, posing a threat that cannot be ignored to food safety and human health. According to the estimates of the World Health Organization, billions of people suffer from food-borne diseases every year in the world, and the probability of food-borne diseases in developed countries is also quite high. On average, one-third of the population is infected with food-borne diseases every year. Foodborne diseases caused by sexual microorganisms accounted for 37.1%. Traditional detection methods include enrichment culture scre...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04C40B40/06
CPCC12Q1/6844C40B40/06C12Q2531/125C12Q2563/107C12Q2545/113Y02A50/30
Inventor 李同祥黄天姿孙会刚汤薇田林王陶李文
Owner XUZHOU UNIV OF TECH
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