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Method for detecting pathogenic shigella by using suspending chip technique

A Shigella, suspension chip technology, applied in biochemical equipment and methods, microbial determination/inspection, fluorescence/phosphorescence, etc., can solve problems such as inability to identify pathogenicity

Inactive Publication Date: 2009-04-08
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

[0015] 70% of the flora in Shigella are pathogenic, and the main detection methods of Shigella, plate culture and immunological methods, can only detect Shigella, but cannot identify whether it is pathogenic

Method used

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  • Method for detecting pathogenic shigella by using suspending chip technique
  • Method for detecting pathogenic shigella by using suspending chip technique
  • Method for detecting pathogenic shigella by using suspending chip technique

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Design and synthesis of primers

[0035] 1) Sequence acquisition: Through genome-wide analysis of pathogenic Shigella, its specific gene Ial was selected as the target sequence, and the gene sequence was obtained from the GenBank public database, numbered AY206439;

[0036] 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;

[0037] 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-Ial-F: 5′-Biotin-CTGGATGGTATGGTGAGGTTT-3′, the downstream primer is IpaH-R: 5′-AGGAGGCCAACAATTATTTCC-3′, and the amplified fragment size is 320bp;

[0038] 4) Primer synthesis: Shanghai Sangong synthesized the down...

Embodiment 2

[0039] Example 2: Design and synthesis of probes

[0040] 1) Sequence acquisition: designing probes in the non-primer region of the amplified fragment;

[0041] 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;

[0042] 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 Ial-Probe: 5′-NH 2 -(CH 2 ) 12 -AATGTCCATCAAAACCCCACTC-3';

[0043] 4) Probe synthesis: Dalian TakaRa synthesized the above probes.

Embodiment 3

[0044] The preparation method of embodiment three suspension chips

[0045] 1. Materials:

[0046] 2. Method results:

[0047] 1. Dilute the amino-modified probe to 1mM (1nanomole / μl) with pure water;

[0048] 2. Shake the stored microspheres for 20 sec;

[0049] 3. Transfer 200μl microspheres to a brown EP tube;

[0050] 4. Collect the microspheres, centrifuge at 8000g for 1-2min;

[0051] 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;

[0052] 6. Add 2μl 1mM probe to the suspended microspheres, shake for 20sec;

[0053] 7. Prepare fresh 10mg / ml EDC (1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride, 1-ethyl-3-3-dimethylaminopropyl carbodiimide), with dH 2 O;

[0054] 8. Add 2.5 μl of newly prepared 10 mg / ml EDC to the microsphere solution and shake;

[0055] 9. Incubate for 30 minutes at room temperature and in the dark;

[0056] 10. Prepare fresh 10m...

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Abstract

The invention relates to a suspension chip used in the detection of pathogenic shigella, and a detection method of the suspension chip which comprises a microsphere carrier and an oligonucleotide probe which is fixed on the chitosan microsphere; wherein, the probe is a segment of DNA sequence of the gene of a specific genic invasion associated locus (invasion associated locus, Ial) screened from the pathogenic shigella. After the genomic DNA of samples to be tested is amplified and marked by utilizing a designed primer, the suspension chip is used for hybridization and whether the pathogenic shigellae is contained in the samples to be tested can be judged according to the crossbred fluorescent intensity. The suspension chip which is high in detection sensitivity can reach the level of the DNA of an fg grade genome, can meet the needs of the detection of clinical samples and environmental samples, is characterized by high specificity, simple operation, low cost, and the like, and can be easily popularized. Furthermore, a PCR reaction system of UNG-Taq enzyme is adopted, which greatly reduces the results of false positive of the PRC.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to the detection of pathogenic shigella 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 pos...

Claims

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Application Information

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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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