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Method for detecting Vibrio cholerae O139 by using suspension chip technology

A Vibrio cholerae and suspension chip technology, which is applied to biochemical equipment and methods, microbiological measurement/testing, fluorescence/phosphorescence, etc., can solve the problems of cumbersome operation, high false negatives, and long time consumption

Inactive Publication Date: 2013-03-20
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The routine detection of Vibrio cholerae is mainly based on the traditional culture method, which is not only cumbersome and time-consuming, but also has a high number of false negatives.

Method used

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  • Method for detecting Vibrio cholerae O139 by using suspension chip technology
  • Method for detecting Vibrio cholerae O139 by using suspension chip technology
  • Method for detecting Vibrio cholerae O139 by using suspension chip technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Design and synthesis of primers

[0034] 1) Sequence acquisition: By analyzing the sequence of the Vibrio cholerae O-antigen gene cluster, the O139 specific gene rfb was selected as the target sequence, and the gene sequence was obtained from the GenBank public database, numbered Y07786;

[0035] 2) Design primers: use Primer Premier 5.0 software to design primers, the relevant parameters are: Tm value 55.0°C-59.0°C, GC value 40.0%-60.0%, PCR product size 100bp-500bp, primer size 22±3bp;

[0036]3) Primer selection: Properly adjust the primers output by the software manually, increase or decrease a few bases, then perform online Blast comparison in GenBank, select primers with high specificity, and perform one of the 5′-end Biotin mark. The upstream primer sequence is O139rfb-F:5'-ATGAAGAAGGATGACCGTATTG-3', the downstream primer is B-O 139rfb-R:5'-Biotin-ACGGAGAAAGTGAGTTTTGCC-3', and the amplified fragment size is 450bp;

[0037] 4) Primer synthesis: Shangh...

Embodiment 2

[0038] Example 2: Design and synthesis of probes

[0039] 1) Sequence acquisition: designing probes in the non-primer region of the amplified fragment;

[0040] 2) design probes: adopt Primer Premier 5.0 software to design probes, select the Hybridization Probes command, design probes on the Anti-sense chain, and the parameters are the same as in Example 1;

[0041] 3) Probe selection: Properly adjust the probes output by the software manually, increase or decrease a few bases, then perform online Blast comparison in GenBank, select probes with high specificity, and perform NH at the 5′ end 2 -(CH 2 ) 12 Modification, the selected probe sequence is O139rfb-Probe:5′-NH 2 -(CH 2 ) 12 -GTGTATGTGGTACCTGGGTTG-3';

[0042] 4) Probe synthesis: Dalian TakaRa synthesized the above probes.

Embodiment 3

[0043] The preparation method of embodiment three suspension chips

[0044] 1. Dilute the amino-modified probe to 1mM (1nanomole / μl) with pure water;

[0045] 2. Shake the stored microspheres for 20 sec;

[0046] 3. Transfer 200μl microspheres to a brown EP tube;

[0047] 4. Collect the microspheres, centrifuge at 8000g for 1-2min;

[0048] 5. Discard the supernatant, add 50μl 0.1M MES (2-[N-Morpholino]ethanesulfonic acid, 2-(N-Morpholino)ethanesulfonic acid) solution pH 4.5, shake for 20sec;

[0049] 6. Add 2 μl of 1mM probe to the suspended microspheres and shake for 20 sec;

[0050] 7. Prepare fresh 10mg / ml EDC (1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride, 1-ethyl-3-3-dimethylaminopropylcarbodiimide) with dH 2 O;

[0051] 8. Add 2.5 μl of newly prepared 10 mg / ml EDC to the microsphere solution and shake;

[0052] 9. Incubate for 30 minutes at room temperature and in the dark;

[0053] 10. Prepare fresh 10mg / ml EDC again with dH 2 O;

[0054] 11. Add ...

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Abstract

The invention relates to a suspended chip and a method for detecting vibrio cholerae O139. The suspended chip comprises a microspheric carrier and an oligonucleotide probe fixed on the carrier, wherein the oligonucleotide probe is a DNA sequence in a vibrio cholerae O139-specific gene rfb screened from gene clusters of O-antigen of vibrio cholerae. A designed primer is used to proliferate and label the genomic DNA of the sample under test, hybridization is performed with the suspended chip, and then whether the sample under test contains vibrio cholerae O139 or not is judged. The suspended chip has high sensitivity, reaches the level of fg genomic DNA, satisfies the requirement of detection on clinical and environmental samples, and is characterized by high specificity, simple operation, low cost and easy generalization. The invention adopts a UNG-taq catalytic PCR system which dramatically decreases the frequency of false-positive outcome in PCR.

Description

technical field [0001] The invention relates to a molecular biology detection chip, in particular to a detection chip for vibrio cholerae O139 using a suspension chip technology. Background technique [0002] Suspension chip technology is a new technology produced by combining gene chip technology and flow cytometry technology. It is a multiple data acquisition and analysis platform. The full name is "Flexible Multiple Analyte Profiling, xMap), also known as Multi-Analyte Suspension Array, organically integrates fluorescently encoded microspheres, laser technology, microfluidic technology, fast signal processing and data analysis systems, which not only guarantees signal quality, but also provides high-throughput new generation molecules Detection technology platform. The biggest difference between the suspension chip and the traditional gene chip is that the traditional gene chip is spotted on a glass slide or a nylon membrane and addressed by coordinate positioning, while...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
CPCY02A50/30
Inventor 王景林赵金银高姗刘艳华康琳
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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