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Reagent kit for detecting vibrio vulnificus by loop-mediated isothermal amplification technology

A loop-mediated isothermal, vibrio vulnificus technology, applied in the field of pathogen diagnosis, can solve problems such as detection errors, achieve the effects of convenient operation, strong specificity, and avoid repeated cultivation

Inactive Publication Date: 2009-01-21
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since Vibrio vulnificus is mainly distributed in seafood and seawater, there are inevitably polymerase chain reaction inhibitors in the test samples, which can often lead to detection errors

Method used

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  • Reagent kit for detecting vibrio vulnificus by loop-mediated isothermal amplification technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: seawater sample 1

[0041] 1. DNA extraction

[0042] Pick suspected colonies isolated from seawater and add them to 0.4mLTE buffer (pH8.0) for shaking and mixing. Add 40 μL of sodium dodecylsulfonate (10%), and then 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 amplifi...

Embodiment 2

[0066] Embodiment 2: clinical isolate sample 1

[0067] 1. DNA extraction

[0068] Pick the colonies and add them to 0.4mL TE buffer (pH8.0) for shaking and mixing. Add 40 μL of sodium dodecylsulfonate (10%), and then 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.

[0069] 2) Loop-mediated isothermal amplification (LAMP)

[0070] Add the following reagents to the PCR reagent tube to make a...

Embodiment 3

[0084] Embodiment 3: clinical sample 2

[0085] 1. DNA extraction

[0086] Pick suspected colonies and add them to 0.4mL TE buffer (pH8.0) for shaking and mixing. Add 40 μL of sodium dodecylsulfonate (10%), and then 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.

[0087] 2) Loop-mediated isothermal amplification (LAMP)

[0088] Add the following reagents to the PCR reagent tube to make a t...

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Abstract

The invention relates to a reagent kit and a method for detecting Vibrio vulnificus by means of a loop-mediated isothermal amplification (LAMP) technology, belonging to the pathogen diagnosis field. The main technical proposal comprises the following steps that by means of the LAMP technology, four high-specificity primers are designed according to six zones of virulence-correlated gene of Vibrio vulnificus, thereby realizing specific detection of Vibrio vulnificus. Compared with the prior method, the method requires simple equipment and operation steps, and has the advantages of quickness, high specificity, strong stability, high sensitivity and the like; moreover, the method has lower detection cost, and is suitable for common clinic and field detection.

Description

technical field [0001] The invention belongs to the field of pathogenic bacteria diagnosis, and the main content is to specifically amplify a virus-correlated gene (virulence-correlated gene) by using loop-mediated isothermal amplification (LAMP) technology, and to treat pathogenic Vibrio vulnificus (Vibrio vulnificus) Do a quick test. The purpose of distinguishing Vibrio vulnificus from similar pathogenic bacteria can be achieved by incubating under isothermal conditions (about 62° C.) for tens of minutes. The method can greatly improve the efficiency of environmental detection and clinical detection of Vibrio vulnificus. Background technique [0002] Vibrio vulnificus is a low-degree halophilic bacterium naturally distributed in the marine environment, which can be isolated from coastal seawater, shellfish and other seafood. There are a considerable number of Vibrio vulnificus infection cases in the United States, Japan, Israel and my country. It can cause a variety of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 黄熙泰郑泽军李永君
Owner NANKAI UNIV
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