Genetic liquid phase chip for joint detection of five drastic pathogenic bacteria and detection method thereof

A detection method, a diagnostic technology, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial determination/inspection, etc.

Inactive Publication Date: 2012-07-04
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These techniques are either sensitive or specific, but are limited by multiple detection capabilities, making it difficult to perform multifactorial screening of suspicious samples

Method used

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  • Genetic liquid phase chip for joint detection of five drastic pathogenic bacteria and detection method thereof
  • Genetic liquid phase chip for joint detection of five drastic pathogenic bacteria and detection method thereof
  • Genetic liquid phase chip for joint detection of five drastic pathogenic bacteria and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: adopt the multiplex PCR reaction of five kinds of bacteria six pairs of primers

[0058] 1. Extraction of nucleic acid in the sample: NaI cracking-glass powder adsorption method to extract nucleic acid.

[0059] 2. Prepare a multiplex PCR reaction system according to the following system, with a total volume of 30 μl:

[0060] 10×PCR buffer: 3μl

[0061] Taq E: 0.4 μl

[0062] dNTPs: 0.6 μl

[0063] BA-1-F, BA-1-R (10μM): 0.3μl each

[0064] BA-2-F, BA-2-R (10μM): 0.3μl each

[0065] YP-F, YP-R (10μM): 0.3μl each

[0066] Bru-F, Bru-P (10μM): 0.36μl each

[0067] FT-F, FT-R (10μM): 0.24μl each

[0068] BP-F, BP-R (10μM): 0.24μl each

[0069] Template DNA: 2μl

[0070] wxya 2 O: add up to 30 μl

[0071] 3. Carry out PCR reaction according to the following reaction conditions

[0072] Pre-denaturation:

[0073] 94°C 10 minutes

[0074] 32 loops:

[0075] 94°C for 30 seconds

[0076] 58°C 30 seconds

[0077] 72°C 40 seconds

[0078] extend: ...

Embodiment 2

[0082] Example 2: Coupling of capture probes to microspheres

[0083] 1. Select coded microspheres No. 044, 042, 032, 025, 034, and 027 respectively, and oscillate the microsphere suspension with a vortex oscillator to mix the microspheres evenly.

[0084] 2. Take the above microspheres about 1.25×10 6 Each was transferred to a centrifuge tube, centrifuged at 14000g for 3-5min, and the supernatant was carefully aspirated.

[0085] 3. Add 50 μl of 0.1 mol / L 2-(n-morpholino)ethanesulfonic acid solution, shake for 20-30 s, and sonicate for 20-30 s to resuspend the microspheres.

[0086] 4. Dilute the synthesized oligonucleotide probe to 0.1 mmol / L with distilled water.

[0087] 5. Add 1 μl of the diluted probe to the microsphere suspension, and add anthrax probe 1, probe 2, and Yersinia pestis probe to the coded microspheres corresponding to 044, 042, 032, 025, 034, and 027, respectively. Brucella probe, tularemia probe, melioidosis probe, shake and mix well.

[0088] 6. Add ...

Embodiment 3

[0097] Example 3: Detection of samples to be tested with the liquid-phase chip of the above-mentioned five kinds of virulent pathogenic bacteria

[0098] 1. Take 3500 of each of the above-mentioned coupled microspheres and mix them, and distribute them in PCR tubes (calculate the corresponding addition amount according to the counting results of the microspheres)

[0099] 2. Add 5-17 μl of the PCR products of the five bacterial mixed templates to each tube to make the final volume 50 μl, and mix well by pipetting.

[0100] Denature at 3.95°C for 10 minutes.

[0101] 4. React at 45-60°C for 10-30 minutes.

[0102] 5. Transfer to a 96-well filter plate and filter to remove unbound PCR products.

[0103] 6. Add 75 μl 4ng / μl SA-PE to each well, incubate at room temperature in the dark for 10 minutes, and filter to remove unbound SA-PE.

[0104] 7. Add 75 μl of the suspension to each well and shake to resuspend the microspheres.

[0105] 8. Check on the machine after the reacti...

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PUM

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Abstract

The invention discloses a genetic liquid phase chip for rapid detection of five drastic pathogenic bacteria of bacillus anthracis, yersinia pestis, brucella bacteria, francisella tularensis and burkholderia pseudomallei. The liquid phase chip for detecting the five drastic pathogenic bacteria has the advantages of large flux, less required sample capacity, strong specificity, high sensitivity, accuracy, high efficiency, and the like.

Description

technical field [0001] The invention relates to a method for the combined detection of five severe pathogenic bacteria of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella and Burkholderia pseudomallei using a gene liquid chip. The detection method of the gene liquid phase chip. Background technique [0002] There are many kinds of potent pathogens that may be used in bioterrorism, including Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella, Burkholderia mallei, Burkholderia pseudomallei, Hemorrhagic fever virus, botulinum toxin and ricin etc. [0003] Existing pathogen detection technologies, such as biosensors or colloidal gold technologies, are all used in the detection of bioterrorism factors. These techniques are either sensitive or specific, but are limited by multiple detection capabilities, making it difficult to perform multifactorial screening of suspicious samples. Suspension array (suspension array), also known as liquid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64C12R1/01C12R1/07
Inventor 王静孙肖红杨宇文海燕刘衡川胡孔新
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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