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Liquid chip for detecting serotypes of four main O antigens of yersinia enterocolon and application thereof

A serotype and small intestine technology, which is applied in the direction of microbial-based methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome work, long cycle, and time-consuming serological identification, etc., and achieve high detection throughput , the effect of short detection time

Active Publication Date: 2021-03-12
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mainly include: the establishment of a serological typing system is cumbersome and takes a long time; the production and quality control of serum is relatively difficult; the experimental process of serological identification takes a long time (2-6 days), etc.

Method used

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  • Liquid chip for detecting serotypes of four main O antigens of yersinia enterocolon and application thereof
  • Liquid chip for detecting serotypes of four main O antigens of yersinia enterocolon and application thereof
  • Liquid chip for detecting serotypes of four main O antigens of yersinia enterocolon and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Design and preparation of serotype-specific primers

[0049] (1) Select Yersinia enterocolitica type O3 wxya gene, O5 type wx gene, O8 wzy gene, and O9-type per Gene is the target gene sequence.

[0050] (2) Import the selected target gene sequences for different serotypes of Yersinia enterocolitica into the primer design software Primer Primer 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 get one upstream and one downstream specific primer for each serotype.

[0051] (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 downstream primer needs to be labeled with a biotin group attached to the 5' end of the DNA sequence.

Embodiment 2

[0053] Design and preparation of serotype-specific probes

[0054] (1) Select Yersinia enterocolitica type O3 wxya gene, O5 type wx gene, O8 wzy gene, and O9-type per Gene is the target gene sequence.

[0055] (2) Import the selected target gene sequences of each serotype into the primer design software Primer Primer 5.0, and set parameters. Among them, only choose the justice chain output mode; Haripin: None; Dimer: None: False Priming: None; Cross Dimer: None. The position of the sequence is within the positions of the upstream and downstream primers in Example 1. Run the program to get 1 specific probe for each serotype.

[0056] (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

[0058] Coupling of specific probes to microspheres (need to be protected from light)

[0059] (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.

[0060] (2) Take 80 microliters of microspheres in a 1.5mL centrifuge tube, centrifuge at 12000 rpm for 2 minutes.

[0061] (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;

[0062] (4) Add 2 μl of probe (placed at room temperature in advance) and 6 μl of freshly prepared 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide at a concentration of 10 mg / mL Hydrochloride solution (EDC), mix well, and incubate at room temperature in the dark for 30 minutes (shake and mix every 15 minutes).

[0063] (5) Add 6 microliters of freshly prepared 10 mg / mL EDC solution, mix well, and incubate at room temperature in the dark for 30 ...

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Abstract

The invention relates to a liquid chip detection method for serotypes of four main pathogenic O antigens of yersinia enterocolon and an application thereof. According to the invention, an O3 type wbbUgene, an O5 type wzt gene, an O8 type wzy gene and an O9 type per gene of yersinia enterocolon are used as target genes, specific primers and probes for each serotype are designed and screened, and acorresponding liquid chip detection method is established. By utilizing the method provided by the invention, molecular biological detection can be accurately, quickly and sensitively carried out onfour main pathogenic O antigen serotype strains of yersinia enterocolon.

Description

technical field [0001] The invention belongs to the technical field of bacterial detection methods, and relates to a liquid-phase chip for detection of four main pathogenic type O antigen serotypes of Yersinia enterocolitica and its application. Background technique [0002] Yersinia enterocolitica is a Gram-negative enteric pathogen. It infects the human body through the fecal-oral transmission route. In addition to causing a variety of gastrointestinal diseases, it can also cause cardiovascular system, respiratory system, bone knot tissue and other diseases, and even sepsis, leading to death. Contamination of food and water sources is an important cause of gastrointestinal enterocolic Yersiniasis, and its transmission routes include human-to-human, human-to-animal, water and food transmission. Yersinia enterocolitica infection has been reported in at least 40 countries around the world, and there have been outbreaks of this bacteria in history in my country, and there are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6837C12Q1/10C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6837C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/143C12Q2563/149C12Q2563/107Y02A50/30
Inventor 郭玺黄敏徐艳丽穆会乾刘斌
Owner NANKAI UNIV
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