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Method for detecting Shigella sonnei by using suspension chip technology

A suspension chip and microsphere technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve the problem of not being able to obtain further information at the species level, and achieve easy promotion, high sensitivity, low cost effect

Inactive Publication Date: 2009-05-27
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

At present, in the mature detection technology of Shigella, only the detection at the genus level can be realized, and further information at the species level cannot be obtained.

Method used

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  • Method for detecting Shigella sonnei by using suspension chip technology
  • Method for detecting Shigella sonnei by using suspension chip technology
  • Method for detecting Shigella sonnei by using suspension chip technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Design and synthesis of primers

[0036] 1) Sequence acquisition: Through the sequence analysis of the O-antigen gene cluster of Shigella sonnei, the specific gene rfc was selected as the target sequence, and the gene sequence was obtained from the GenBank public database, numbered AF294823.1;

[0037] 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;

[0038] 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 B-S.s rfc-F: 5′-Biotin-GGATAGCCGAGCAGGAATA-3′, the downstream primer is S.s rfc-R: 5′-CCCTAACTGAGCCGAATAAGA-3′, and the amplified fragment size is 392bp;

[0039] 4) Primer synthesis: S...

Embodiment 2

[0040] Example 2: Design and synthesis of probes

[0041] 1) Sequence acquisition: designing probes in the non-primer region of the amplified fragment;

[0042] 2) design probes: adopt Primer Premier 5.0 software to design probes, select the HybridizationProbes command, design probes on the Anti-sense chain, and the parameters are the same as in Example 1;

[0043] 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 Modified, the selected probe sequence is S.s rfc-Probe: 5′-NH 2 -(CH 2 ) 12 -CGTTCAAGACGAGTGCCTAT-3';

[0044] 4) Probe synthesis: Dalian TakaRa synthesized the above probes.

Embodiment 3

[0045] Example 3 Preparation method of suspension chip

[0046] 1. Dilute the amino-modified probe to 1mM (lnanomole / μl) with pure water;

[0047] 2. Shake the stored microspheres for 20 sec;

[0048] 3. Transfer 200μl microspheres to a brown EP tube;

[0049] 4. Collect the microspheres, centrifuge at 8000g for 1-2min;

[0050] 5. Discard the supernatant, add 50 μl 0.1M MES (2-[N-Morpholino]ethanesulfonic acid, 2-(N-Morpholino)ethanesulfonic acid) solution pH4.5, shake for 20sec;

[0051] 6. Add 2μl 1mM probe to the suspended microspheres, shake for 20sec;

[0052] 7. Prepare fresh 10mg / ml EDC (1-ethyl-3-[3dimethylaminopropyl]carbodiimidehydrochloride, 1-ethyl-3-3-dimethylaminopropylcarbodiimide), with dH 2 O;

[0053] 8. Add 2.5 μl of newly prepared 10 mg / ml EDC to the microsphere solution and shake;

[0054] 9. Incubate for 30 minutes at room temperature and in the dark;

[0055] 10. Prepare fresh 10mg / ml EDC again with dH 2 O;

[0056] 11. Add 2.5 μl of newly prep...

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Abstract

The invention relates to a suspension chip for Shigella sonnei detection, and a detection method thereof. The suspension chip comprises a microsphere carrier and an oligonucleotide probe fixed on the carrier, wherein the oligonucleotide probe is a section of DNA sequence in Shigella sonneispecific gene rfc screened from Shigella O-antigen gene cluster. The method comprises the steps of utilizing a designed primer to amplify and mark genomic DNA of a sample to be detected, utilizing the suspension chip to perform hybridization and judging whether the sample to be detected contains the Shigella sonnei according to hybridization fluorescence intensity. The suspension chip is high in detection sensitivity, can reach the level of fg-grade genomic DNA, can meet the detection requirement of clinical samples and environmental samples, has the characteristics of high specificity, simple operation, low cost and the like, and is easy to popularize. In addition, the detection method adopts a UNG-Taq enzyme PCR reaction system, thereby greatly reducing false positive results of PCR.

Description

technical field [0001] The invention relates to a molecular biology detection chip, in particular to a method for detecting Shigella sonnei by 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 SuspensionArray, 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 molecular 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 Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
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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