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A liquid chip for detecting Bacillus cereus and its application

A Bacillus cereus and chip technology, applied in the direction of microorganism-based methods, microorganism measurement/inspection, DNA/RNA fragments, etc., can solve the problems of incomplete antiserum, long cycle, time-consuming, etc., and achieve high detection throughput , the effect of short detection time

Active Publication Date: 2022-05-10
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mainly include: 1) The work of establishing a serological typing system is cumbersome and the cycle is long; 2) The production and quality control of antisera are relatively difficult; 3) Many antisera are produced and stored by only a few units in the world, and are used for identification. Antiserum is severely incomplete; 4) The experimental process of serological identification takes a long time (2-6 days)

Method used

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  • A liquid chip for detecting Bacillus cereus and its application
  • A liquid chip for detecting Bacillus cereus and its application
  • A liquid chip for detecting Bacillus cereus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Design and preparation of 8 specific primers for Bacillus cereus

[0076] (1) Select the wzm gene of 8 species of Bacillus cereus as the target gene sequence.

[0077] (2) Import the selected target gene sequences for different Bacillus cereus into the primer design software PrimerPrimier 5.0, and set parameters. Among them, select the sense strand and complementary strand output mode; the sequence amplification length is 150-350bp; Haripin: none; Dimer: none: False Priming: none; Cross Dimer: none. Run the program to obtain a pair of specific primer sequences for the sense strand and the antisense strand of each serotype.

[0078] (3) Send the designed primer sequences to Thermo Fisher Scientific (China) Co., Ltd. for DNA synthesis and PAGE purification for future use. Among them, the synthesis of the antisense strand primer needs to be labeled with a biotin group attached to the 5' end of the DNA sequence.

Embodiment 2

[0080] Design and preparation of specific probes for Bacillus cereus

[0081] (1) Select the wzm gene of Bacillus cereus G6235, G6206, G6239, G2724, G6211, G6217, G6207, G6214 as the target gene sequence. .

[0082] (2) Import the selected target gene sequences for different Bacillus cereus into the primer design software PrimerPrimier 5.0, and set parameters. Wherein, only the sense strand output mode is selected; Haripin: None; Dimer: None: FalsePriming: None; Cross Dimer: None, and the position of the sequence is within the position of the sense strand and antisense strand primers in Example 1. Run the program to get 1 specific probe for each serotype.

[0083] (3) Send the designed probe sequence to Thermo Fisher Scientific (China) Co., Ltd. for DNA synthesis. At the same time, 12 carbon atoms are connected to the 5' end of the sequence as a connecting arm, and the last carbon An amino group is connected to the end of the atom, purified by PAGE, and set aside.

Embodiment 3

[0085] Coupling of specific probes to microspheres (need to be protected from light)

[0086] (1) Suspend the microspheres on the vortex at the highest speed for 30 s, check the numbers of the microspheres and probes, and mark them.

[0087] (2) Take 80 microliters of microspheres in a 1.5mL low adsorption centrifuge tube, centrifuge at 12000 rpm for 5 minutes.

[0088] (3) Discard the supernatant, resuspend with 10 microliters of 0.1 mol / L 2-(N-morpholine)ethanesulfonic acid solution (MES) (pH4.5), and vortex thoroughly to disperse the microspheres;

[0089] (4) Add 4 microliters of probe (placed at room temperature in advance) and 2.5 microliters of freshly prepared 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt at a concentration of 10 mg / mL salt solution (EDC), mix well, and incubate at room temperature in the dark for 30 minutes (shake and mix every 15 minutes).

[0090] (5) Add 2.5 microliters of freshly prepared 10 mg / mL EDC solution, mix well, and incubate at roo...

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Abstract

The present invention provides DNA sequences for detecting 8 kinds of Bacillus cereus, DNA probes connected to magnetic microspheres marked with different fluorescent dyes, including: Bacillus cereus G6235, G6206, G6239, G2724, G6211, A DNA fragment selected from G6217, G6207, and G6214 respectively. Combining the Bio-Plex 200 suspension chip system of Bio-Rad, the present invention establishes a set of suspension chip detection system and its detection method for detecting 8 kinds of Bacillus cereus, which fills the gap in the detection of wax samples by molecular biology methods. The technical gap in the detection of Bacillus is of great significance for the clinical identification and epidemiological monitoring of this important pathogenic bacteria.

Description

technical field [0001] The invention belongs to the technical field of bacterial detection methods, and relates to a liquid phase chip used for detection of 8 kinds of Bacillus cereus and its application. Background technique [0002] Bacillus cereus is a facultative anaerobic Gram-positive bacterium that widely exists in soil, water, air, and animal intestines, and is closely related to humans. Because Bacillus cereus is a common bacterium that causes food poisoning, it mainly contaminates starch products, milk and dairy products. Under certain conditions, it can produce enterotoxin, which can cause vomiting and diarrhea food poisoning in humans. [0003] In the entry-exit inspection and quarantine industry standard (SNT0176-2013) issued by my country in 2013 and the national standard (GB4789.14-2014) issued in 2014, the detection of Bacillus cereus in food is based on the pure culture of bacteria. Based on the method of biochemical identification or colony counting, the in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6837C12Q1/04C12N15/11C12R1/085
CPCC12Q1/689C12Q1/6837C12Q2600/16C12Q2563/143C12Q2563/149C12Q2563/107C12Q2531/113C12Q2537/143
Inventor 刘斌王婧郭玺穆会乾
Owner NANKAI UNIV
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