Kit for detecting guinea pig aeromonas by utilizing Loop-mediated isothermal amplification technique

A ring-mediated isothermal technology for Aeromonas caviae, applied in the field of pathogenic bacteria diagnosis, can solve the problems of lack of rapid and accurate detection methods for pathogenic bacteria, and achieve the effects of convenient operation, strong specificity, and simple equipment

Inactive Publication Date: 2008-08-06
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Domestic case reports related to Aeromonas guinea pigs are not uncommon, bu...

Method used

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  • Kit for detecting guinea pig aeromonas by utilizing Loop-mediated isothermal amplification technique
  • Kit for detecting guinea pig aeromonas by utilizing Loop-mediated isothermal amplification technique
  • Kit for detecting guinea pig aeromonas by utilizing Loop-mediated isothermal amplification technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Patient Sample 1

[0041] 1. DNA extraction

[0042] Pick suspected colonies and add them to 0.4mLTE buffer (pH8.0) for shaking and mixing. Add 10 μL of proteinase K solution and incubate at 55°C for 1-2 hours. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 8000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (3mol / L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant, Wash once with 70% cold ethanol, centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 50 μL TE solution, and store at -20°C.

[0043] 2) Loop-mediated isothermal amplification (LAMP)

[0044] Add the following reagents to the PCR reagent tube to make a total volume of 25 μL:

[0045] Composition Final concentration...

Embodiment 2

[0070] Example 2: Patient Sample 2

[0071] 1. DNA extraction

[0072] Pick suspected colonies and add them to 0.4mLTE buffer (pH8.0) for shaking and mixing. Add 10 μL of proteinase K solution and incubate at 55°C for 1-2 hours. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 8000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (3mol / L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant, Wash once with 70% cold ethanol, centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 50 μL LTE solution, and store at -20°C.

[0073] 2) Loop-mediated isothermal amplification (LAMP)

[0074] Add the following reagents to the PCR reagent tube to make a total volume of 25 μL:

[0075] 10× Reaction Buffer (2.5 μL)...

Embodiment 3

[0088] Example 3: Patient Sample 3

[0089] 1. DNA extraction

[0090] Pick suspected colonies and add them to 0.4mLTE buffer (pH8.0) for shaking and mixing. Add 10 μL of proteinase K solution and incubate at 55°C for 1-2 hours. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 8000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (3mol / L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant, Wash once with 70% cold ethanol, centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 50 μL LTE solution, and store at -20°C.

[0091] 2) Loop-mediated isothermal amplification (LAMP)

[0092] Add the following reagents to the PCR reagent tube to make a total volume of 25 μL:

[0093] 10× Reaction Buffer (2.5 μL)...

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Abstract

The invention relates to a reagent kit and a detecting method for using loop-mediated isothermal amplification (LAMP) technique to detect aeromonas caviae, which belongs to the pathogen diagnosis field. The main technical scheme of the invention comprises: utilizing the LAMP technique, designing four pairs of high specificity primers aiming at six zones of 16s and 23s ribosome RNA gene transcription spacer region, and achieving the purpose for detecting the aeromonas caviae with specificity. Compared with a traditional method, equipment and operational steps which are needed in the method are simple, the method has the advantages of rapidness and high specificity, and the detecting cost is lower, which is suitable for common clinics and field detections.

Description

technical field [0001] The invention belongs to the field of diagnosis of pathogenic bacteria, and the main content is to specifically amplify the intertranscriptional regions of 16S and 23S ribosomal RNA genes by using loop-mediated isothermal amplification (loop-mediateddisotherm amplification LAMP) technology to treat pathogenic Aeromonas murine cells. bacteria (Aeromonas caviae) for rapid detection. The purpose of distinguishing Aeromonas caviae from similar pathogenic bacteria can be achieved by incubating for tens of minutes under isothermal conditions (about 65°C). The method can greatly improve the efficiency of clinical diagnosis of Aeromonas caviae. Background technique [0002] Aeromonas caviae is widely distributed in the natural environment, especially in various water bodies. The isolation of Aeromonas caviae from fresh water, brackish water, tap water and soil, as well as animal and human feces has been reported in the literature. This bacterium is related ...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68
Inventor 黄熙泰魏晓娜郑泽军
Owner NANKAI UNIV
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