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Method for detecting Shigella by using suspension chip technology

A Shigella, suspension chip technology, applied in biochemical equipment and methods, microbial determination/inspection, fluorescence/phosphorescence, etc., can solve the problem of difficult to control false positive and false negative rates, immature technology, cross-contamination, etc. question

Inactive Publication Date: 2009-05-13
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 detection efficiency of the gene chip method is high, but the technology is immature, the false positive and false negative rates are difficult to control, and the cost is high, and it is still in the research stage
Ordinary PCR technology is mature, and a large number of scholars have reported that this technology is applied to the detection of pathogenic bacteria, but it is prone to false negatives and cross-contamination, so it has not been widely used in clinical practice.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Design and synthesis of primers

[0053] 1) Sequence acquisition: Through genome-wide analysis of Shigella, the specific gene IpaH was selected as the target sequence, and the gene sequence was obtained from the GenBank public database, numbered SHFIPAHX;

[0054] 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;

[0055] 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-IpaH-F: 5′-Biotin-CTTGACCGCCTTTCCGATAC-3′, the downstream primer is IpaH-R: 5′-ACTCCCGACACGCCATAGA-3′, and the amplified fragment size is 421bp;

[0056] 4) Primer synthesis: Shanghai Sangong synthesized downstream primer ...

Embodiment 2

[0057] Example 2: Design and synthesis of probes

[0058] 1) Sequence acquisition: designing probes in the non-primer region of the amplified fragment;

[0059] 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;

[0060] 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 Ipah-Probe: 5′-NH 2 -(CH 2 ) 12 -CGGAGATTGTTCCATGTGAGC-3';

[0061] 4) Probe synthesis: Dalian TakaRa synthesized the above probes.

Embodiment 3

[0062] Example 3: Suspension Chip Rapid Detection Method for Shigella

[0063] 1) TIANamp Bacteria DNA kit was used to extract the genomic DNA of the sample to be tested.

[0064] 2) PCR amplification of the target fragment, the reaction conditions are as follows:

[0065] Table 2 PCR reaction system

[0066]

[0067] The PCR reaction program was: denaturation at 94°C for 3 min; 35 cycles of 30 sec at 94°C, 30 sec at 53°C, 40 sec at 72°C; finally, extension at 72°C for 3 min.

[0068] 3) Hybridization: Hybridization solution composition: 33.3 μl 1.5×TMAC (tetramethylammonium chloride, tetramethylammonium chloride), 5 μl of the above PCR product, 5000 probe-coupled microspheres were added, and 1×TE to make up the volume to 50 μl. The hybridization solution was denatured at 95°C for 4 minutes, and hybridized at 52°C for more than 15 minutes.

[0069] 4) Detection: Transfer the above-mentioned hybridized solution to a 96-well filter plate, collect the microspheres by vacuum...

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Abstract

The invention relates to a suspension chip for detecting shigella and a detecting method thereof. The suspension chip comprises a microsphere carrier and an oligonucleotide probe fixed on the carrier, wherein the oligonucleotide probe is a fragment of DNA sequence in specific gene invasive plasmid antigen H screened from the shigella. The detecting method comprises the following steps: after a genome DNA in a sample to be detected is amplified and labeled by a designed primer, using the suspension chip to hybridize the genome DNA, and judging whether the sample to be detected contains the shigella according to hybridized fluorescence intensity. The suspension chip has high detection sensitivity which can reach a level of 1.18fg genome DNA (1fg corresponds to 2 numbers of bacteria), so the method can fully meet detection requirements of clinical samples and environmental samples, has the characteristics of high specificity, simple operation, low cost and the like, and is easy to popularize. And the method adopts a PCR reaction system of UNG-Taq enzyme to reduce PCR false positive results greatly.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to the detection of shigella by applying the suspension chip technology. Background technique [0002] Humans are highly sensitive to Shigella, and only less than ten bacteria can cause human infection, so it is highly contagious and harmful. Shigella can be transmitted through water and food, causing diarrhea and dysentery in humans. According to relevant domestic data, Shigella ranks first among the pathogens of infectious diarrhea in my country; data from the US National Reporting Disease Surveillance System and Public Health Test Information System show that there are as many as 448,240 cases of Shigella every year 70 cases; the WHO annual report pointed out that there are as many as 150-250 million cases of bacterial diarrhea in Asia, Africa and Latin America, and 650,000 deaths. In 2002, Order No. 25 and No. 26 of the General Administration of Quality Su...

Claims

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Application Information

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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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