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A kind of Pseudomonas aeruginosa detection kit and its application

A Pseudomonas aeruginosa, detection kit technology, applied in the direction of determination/inspection of microorganisms, microorganisms, microorganism-based methods, etc., can solve problems such as unsatisfactory reliability and repeatability, and achieve short detection time and specificity. The effect of strong, high-confidence detection

Active Publication Date: 2018-11-27
XIAMEN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For Pseudomonas aeruginosa, which has important research significance in hygiene and pathology, researchers at home and abroad have been working on the screening of target genes and primer sequences identified by PCR for many years. Commonly used sequences include 16S rDNA, 16S-23S rDNA ITS , oprI, oprL, fliC, gyrB, ecfX, etc., but their reliability and repeatability are not ideal, different reports have different opinions, and there are certain doubts in actual operation

Method used

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  • A kind of Pseudomonas aeruginosa detection kit and its application
  • A kind of Pseudomonas aeruginosa detection kit and its application
  • A kind of Pseudomonas aeruginosa detection kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A Pseudomonas aeruginosa detection kit, comprising the upstream primer of the sequence shown in SEQ ID 01, the downstream primer of the sequence shown in SEQ ID02, selective enrichment solution, PCR reaction reagent, positive control, negative control and blank For comparison, the ratio of each component in the PCR reaction system of the kit is as follows: 25 μL of the PCR reaction system includes:

[0029]

[0030]

[0031] The amplification temperature program of the above PCR reaction is as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 60°C for 30 s, extension at 72°C for 15 s, 35 cycles; extension at 72°C for 7 min.

[0032] The formula ratio of the above selective enrichment solution is: 1L of the selective enrichment solution contains tryptone 10g, sodium chloride 10g, yeast extract 5g, cetyltrimethylammonium bromide 0.2g and naphthyridone Acid 0.015g.

[0033] The method for detecting using the above-mentioned Ps...

Embodiment 2

[0037] Example 2: Verification of the specificity of the detection primers in the present invention.

[0038]In this example, 6 strains of Pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC27853, ATCC15442, ATCC9027, CGMCC1.10274, CGMCC1.10452, XMZJ~1 (separated from the laboratory and confirmed as Pseudomonas aeruginosa by VITEK2Compact identification), 19 strains of other bacteria of the genus Pseudomonas [P.azotoformans CGMCC1.1792, P.mucidolens CGMCC1.1795, Pseudomonas alcaligenes ) CGMCC 1.3361, Pseudomonas putida (P.putida) CGMCC 1.3301, Pseudomonas pseudoalcaligenes (P.pseudoalcaligenes) CGMCC 1.2935, Pseudomonas stutzeri (P.stutzeri) CGMCC1.3340, Mendoza Pseudomonas (P.mendocina) CGMCC 1.2965, Pseudomonas asplenia (P.asplenii) CGMCC 1.4995, Pseudomonas chlororaphis (P.chlorophis) CGMCC 1.2887, Pseudomonas chicory (P.cichorii ) CGMCC 1.4934, Pseudomonas flavescens (P.flavescens) CGMCC1.6138, Pseudomonas citronellolis (P.citronellolis) CGMCC1.6143, Pseudomonas jesseni...

Embodiment 3

[0055] Example 3: Detection of Pseudomonas aeruginosa in bottled drinking purified water.

[0056] This example uses the specific primers and PCR method of Example 1 of the present invention to detect 10 parts of commercially available bottles and barrels of drinking purified water, and compares the parallel detection results of traditional detection methods (GB 19298-2014) to verify the present invention The reliability of the established detection method.

[0057] The primers and PCR method used are the same as in Example 1.

[0058] The main testing instruments used:

[0059] Milliflex~plus microbial filtration system (Millipore, Germany), 250 mL filter cup with 0.45 μm pore size filter membrane, micropipette (10 μL, 100 μL, 1000 μL, Eppendorf), centrifuge (5424R, Eppendorf, Germany), gradient PCR amplification Multiplier (Veriti, American ABI Company), electrophoresis instrument (Tanon EPS 300, Shanghai Tianneng Technology Co., Ltd.), gel imager (Tanon 3500, Shanghai Tia...

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Abstract

The invention discloses a pseudomonas aeruginosa detection kit and an application thereof. The kit comprises upstream primers including a sequence indicated by SEQ ID 01, downstream primers including a sequence indicated by SEQ ID 02, a selective bacterial enriching liquid, a PCR reagent, a positive contrast, a negative contrast and a blank. The specific primers in the kit are designed for o-antigen acetylase gene of pseudomonas aeruginosa, and effectively distinguish between the pseudomonas aeruginosa and common pathogenic bacteria ( including other 19 species of pseudomonas ). The quick, highly reliable detection for pseudomonas aeruginosa is accomplished.

Description

technical field [0001] The invention belongs to the technical field of pathogenic microorganism detection, and in particular relates to a detection kit for Pseudomonas aeruginosa and an application thereof. Background technique [0002] Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is an obligate aerobic, non-fermenting Gram-negative bacillus. The bacterium is widely distributed in humid environments such as water, air, normal human skin, respiratory tract and intestinal tract, etc. Cause sepsis and bacteremia, which can lead to death in severe cases. [0003] In view of its serious threat to human health, strict "no detection" limits have been imposed on Pseudomonas aeruginosa in drinking water, natural mineral water, cosmetics and other products at home and abroad. On May 24, 2015, my country implemented a new standard for packaged drinking water "GB 19298-2014", in which Pseudomonas aeruginosa was also included in the microbial indicators, limited to 5 s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/04C12R1/385
CPCC12Q1/689C12Q2600/158
Inventor 刘舟谢雪钦
Owner XIAMEN MEDICAL COLLEGE
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