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Multiple detection method for field screening of toxicogenic Pseudomonas aeruginosa

A Pseudomonas aeruginosa, multiple detection technology, applied in the field of molecular biology, can solve the problems that the analysis and identification of amplification products need to be improved, and the identification of molecular weight target products cannot be distinguished, and achieves shortened detection time, high sensitivity, and improved detection efficiency. Effect

Inactive Publication Date: 2017-09-15
FUDEAN OF BEIJING TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, because the LAMP amplification product presents a ladder-like band in ordinary electrophoresis, multiple LAMP products cannot be identified by ordinary electrophoresis to distinguish the molecular weight of the target product. In previous reports, more tedious enzyme digestion or sequencing methods were often used. The method to identify the amplified product, the analysis and identification of the amplified product still needs to be improved

Method used

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  • Multiple detection method for field screening of toxicogenic Pseudomonas aeruginosa
  • Multiple detection method for field screening of toxicogenic Pseudomonas aeruginosa
  • Multiple detection method for field screening of toxicogenic Pseudomonas aeruginosa

Examples

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Embodiment 1

[0037] The concrete detection method of the present embodiment is:

[0038] 1. Extraction of sample DNA

[0039] 1.1 The standard strains of Pseudomonas aeruginosa were inoculated in the nutrient broth and cultured at 36°С±1°С for 18 hours.

[0040] 1.2 Use the bacterial genome DNA extraction kit to extract the DNA of the above culture products respectively, and use it as a template for detection.

[0041] 1.3 Mix the two sets of DNA extracts in an equal volume ratio as a template for the detection of mixed target bacteria.

[0042] 2. Multiple LAMP reactions

[0043] 2.1 Artificially synthesized labeled primer sets for the internal standard gene and two toxin genes of Pseudomonas aeruginosa respectively.

[0044] 2.2 Multiple LAMP reaction system

[0045] The composition of the reaction system includes: (1) primer mixture: outer primer F 3 and B 3 0.3 μmol / L each, 2.3 μmol / L each for internal primers FIP and BIP; (2) Reaction mixture: 3mol / L betaine, 0.3mmol / L MgSO 4 , 1...

Embodiment 2

[0057] Take 63 samples of river water, take 2ml of sample solution to extract the genome and perform multiple LAMP amplification, then use colloidal gold test strips to detect multiple LAMP products, and read the test results within 5 minutes. In the case of color development of the quality control line: the detection lines T3, T2 and T1 are not colored, indicating that there is no Pseudomonas aeruginosa in the sample; the detection line T3 is colored, and T2 and T1 are not colored, indicating that there is no color in the sample There are Pseudomonas aeruginosa that does not secrete toxin ExoU and ExoS genes; detection lines T3 and T2 develop color, and T1 does not develop color, indicating that there are Pseudomonas aeruginosa that secrete toxin ExoS but not ExoU genes in the sample; detection line T3 and T1 color development, T2 no color development, indicating that there is Pseudomonas aeruginosa that secretes the toxin ExoU but not the ExoS gene in the sample; the detectio...

Embodiment 3

[0059] Take 78 samples of drinking water, take 2ml of sample solution to extract the genome and perform multiple LAMP amplification, then use colloidal gold test strips to detect multiple LAMP products, and read the test results within 5 minutes. In the case of color development of the quality control line: the detection lines T3, T2 and T1 are not colored, indicating that there is no Pseudomonas aeruginosa in the sample; the detection line T3 is colored, and T2 and T1 are not colored, indicating that there is no color in the sample There are Pseudomonas aeruginosa that does not secrete toxin ExoU and ExoS genes; detection lines T3 and T2 develop color, and T1 does not develop color, indicating that there are Pseudomonas aeruginosa that secrete toxin ExoS but not ExoU genes in the sample; detection line T3 and T1 color development, T2 no color development, indicating that there is Pseudomonas aeruginosa that secretes the toxin ExoU but not the ExoS gene in the sample; the detec...

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Abstract

The invention relates to a multiple detection method for field screening of toxicogenic Pseudomonas aeruginosa and belongs to the field of molecular biology. The method includes LAMP (loop-mediated isothermal amplification) primer design, establishment of a multiple LAMP reaction system, and detection of multiple LAMP products by multiple colloidal gold test strips. In the LAMP primer design, the software Primer Explorer V4 is used to design four LAMP primers for each of 6 sites of each of endogenous reference gene ecfX, toxin gene ExoU and toxin gene ExoS of Pseudomonas aeruginosa; the four LAMP primers include two outer primers F3 and B3 and two inner primers FIP and BIP. The multiple LAMP detection technique used herein combines with the means of multiple colloidal gold test strip detection, allowing the endogenous reference gene and toxin genes of Pseudomonas aeruginosa to be detected fast; the method has the advantages of high speed, good sensitivity, high efficiency, low cost, and the like.

Description

technical field [0001] The invention relates to a multiple detection method for on-site screening of toxin-producing Pseudomonas aeruginosa, belonging to the field of molecular biology. Background technique [0002] Pseudomonas aeruginosa is an important opportunistic pathogen that is widely distributed in soil, water, and surfaces of animals and plants. Pseudomonas aeruginosa can infect burnt or immunocompromised patients, and can cause more serious injury and even death in cystic fibrosis patients. Pseudomonas aeruginosa can secrete some pathogenic factors, which will bind to specific sites in the host body, making it highly pathogenic. Among them, the main pathogenic factor is the type III secretion system T3SS. Pseudomonas aeruginosa can directly inject virulence proteins into the host cells through its T3SS system, interfere with the signal transduction of the host cells, change the functions of the host cells, and create effective An environment conducive to the surv...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6804C12Q1/6844C12Q2537/143C12Q2531/119
Inventor 罗云波许文涛黄昆仑陈玉婷程楠徐瑗聪翟百强
Owner FUDEAN OF BEIJING TECH
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