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Method for quick trace synchronous detection of bacteria

A technology for synchronous detection and bacteria, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., can solve problems such as multi-materials, and achieve the effect of synchronous detection

Inactive Publication Date: 2007-01-24
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the antibody capture method UPPCR for detection requires more materials

Method used

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  • Method for quick trace synchronous detection of bacteria
  • Method for quick trace synchronous detection of bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0014] Use the direct thermal denaturation method to prepare the template, inoculate the bacteria in LB medium, and culture them on a shaker at 28°C for 12-18 hours as seed bacteria, and then inoculate them in the same medium as above at a ratio of 1:50 (bacteria:medium). Incubate on a shaking table at 28-37°C for 12-13 hours; take 0.5ml of the bacterial solution into a sterile 1.5ml centrifuge tube, centrifuge at 3,500 rpm for 20min, discard the supernatant; wash the precipitate with distilled water once, and add 0.8ml of sterile double-distilled water , mix well, boil in 100℃ water bath for 10min; put the centrifuge tube after water bath in 0℃ ice bath to cool, then centrifuge at 12,000rpm for 10min, absorb 15~30μl (25μl PCR reaction system takes 15μl, 50μl PCR reaction system takes 15μl, 50μl PCR reaction system takes 30 μl) supernatant was used as a PCR reaction template.

[0015] Add 2.5 μl 10× buffer, 0.5 μl dNTP (nucleotide mixture, each 10 mM), 0.7 μl MgCl in order 2 ...

Embodiment 2

[0018] Similar to Example 1, the difference is that the template is prepared by thermal denaturation after antibody capture, and antibodies (such as type and group-specific antibodies) that can react with various bacteria are diluted with pH9.6, 0.01mol / L carbonic acid buffer Coat each well of a 96-well enzyme-linked reaction plate (1-10 μg / ml), 50 μl per well, and store in a refrigerator at 4°C overnight; then use pH 7.4, 0.01mol / L phosphate-Tween buffer (PBS- T) Wash the plate 3 times, each time for 3-5 minutes; add 50 μl of 10% calf serum to each well, block at 36-38°C for 1 hour, and shake dry; add 20-40 μl of bacterial liquid culture solution to each well (add 20 μl , 50 μl PCR reaction system plus 40 μl), incubate at 36-38°C for 1-2 hours, wash the plate 5 times with PBS-T washing solution, 3-5 minutes each time. Add 20-40 μl (consistent with the volume of the added bacterial solution) sterile double-distilled water to each well, heat in a boiling water bath for 8-10 min...

Embodiment 3

[0020]Bacterial template preparation: using Pseudomonas fluorescens, Vibrio anguillarum, Vibrio fluvialis, Aeromonas hydrophila, Providencia rettii rettgeri), Aeromonas sobria (Aeromonas sobria), the strains preserved by the applicant. The above-mentioned bacteria were inoculated separately and mixed in LB medium, a total of 7 tubes were cultured on a shaker at 28°C for 12-18 hours, and used as seed bacteria. Then inoculate the above-mentioned same culture medium at a ratio of 1:50 (bacteria:medium), culture on a shaking table at 28°C for 12-13 hours; take 0.5ml of the bacteria solution into a new sterile 1.5ml small centrifuge tube, and run at 3,500 rpm Centrifuge for 20 minutes, discard the supernatant; wash the precipitate with distilled water once, add 0.8ml of sterile double distilled water, mix well, boil in a water bath at 100°C for 10 minutes; put the small centrifuge tube after the water bath in an ice bath at 0°C to cool, Then centrifuge at 12,000 rpm for 10 min, an...

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Abstract

The present invention relates to a fast synchronous inspection method of micro content bacteria, which comprises: 1) mould board preparation, 2) PCR augmentation, the augmentation guide object is the general guide object of bacteria ribosomal ribonucleic acid gene, 3) gel electrophoresis of denaturization gradient, i.e. gel electrophoresis of denaturization gradient for augmentation outgrowth to determine sorts. The augmentation of 16S rRNA gene segment of bacteria is realized through UPPCR technology, obtaining all corresponding bacteria gene segment sequence information, and then DGGE is employed to appraise the augmentation outgrowth of mixed bacteria, so as to confirm the inspected bacteria. The invention provides a fast synchronous inspection method of micro content bacteria by combining the technologies of UPPCY and DGGE, which achieves the advantages of micro content, fast and synchronous inspection for mixed bacteria, especially pathogenic bacteria.

Description

technical field [0001] The invention relates to a method for rapid and synchronous detection of microscopic bacteria, especially pathogenic bacteria. Background technique [0002] Pathogenic bacteria seriously endanger people's health and industrial and agricultural production. To control the disease caused by it, it should be diagnosed in time to take corresponding preventive measures. In recent years, PCR (polymerase chain reaction) technology has been applied to the detection of pathogenic bacteria. These detection techniques can be divided into two categories, namely PCR with specific primers and PCR with non-specific primers. The former has different primer sequences depending on the target bacteria, and different primer sequences may require different amplification conditions. If the bacteria to be tested are unknown, it is difficult to achieve the purpose of rapid and specific detection (Peng X X, Zhang J Y, Wang S Y, et al. al, Immuno-capture PCR for Detection of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 彭宣宪纪念念彭博王三英
Owner XIAMEN UNIV
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