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Primers for detecting pseudomonas aeruginosa and ExoY in water, kit and method

A technology for detecting Pseudomonas aeruginosa and water, applied in biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve the problems of Pseudomonas aeruginosa and ExoY gene detection that have not been reported, and achieve good industry The effect of improving the outlook, shortening the detection time, and shortening the detection cycle

Inactive Publication Date: 2018-02-02
INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, there are few reports on the analysis of copy number based on the droplet digital PCR platform, let alone the detection of Pseudomonas aeruginosa and ExoY genes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The present invention detects the primers of Pseudomonas aeruginosa and ExoY gene in water, and the sequences are respectively:

[0044] Upstream primer P.AF: TGGTAGTCCACGCCGTAAA

[0045] Downstream primer P.AR:CAGACTGCGATCCGGACTACG

[0046] Probe P.AP: TCGACCGCCTGGGGAGTACG

[0047] Upstream primer ExoYF: GTCTTCTACAGGATCAGCCGCGC

[0048] Downstream primer ExoYR: TGCGCATCAGACCTTACGTT

[0049] Probe ExoYP: GGCAACTGGTGGACGCCCTC.

Embodiment 2

[0051] The present invention is a kit for detecting Pseudomonas aeruginosa and ExoY gene in water, wherein the 20 μL reaction system includes the following components:

[0052] Among them, 10.0 μL of 2×ddPCR Super Mix, 1.0 μL of forward and reverse primers for Pseudomonas aeruginosa and ExoY, 1.0 μL of each probe, and 4.0 μL of DNA template.

[0053] Wherein the primer sequences are as follows:

[0054] Upstream primer P.AF: TGGTAGTCCACGCCGTAAA

[0055] Downstream primer P.AR:CAGACTGCGATCCGGACTACG

[0056] Probe P.AP: TCGACCGCCTGGGGAGTACG

[0057] Upstream primer ExoYF: GTCTTCTACAGGATCAGCCGCGC

[0058] Downstream primer ExoYR: TGCGCATCAGACCTTACGTT

[0059] Probe ExoYP: GGCAACTGGTGGACGCCCTC.

Embodiment 3

[0061] The present invention detects Pseudomonas aeruginosa and ExoY gene method in water, comprises the following steps:

[0062] A. Extract sample DNA;

[0063] B. Add each reaction component to the 20.0 μl reaction system as in Example 2, then add 70.0 μl mineral oil, transfer to the droplet generator to automatically generate droplets after mixing;

[0064] C. Carefully transfer all the generated micro-droplets to the PCR reaction tube of the 96-well reaction plate; then seal the 96-well reaction plate on a film sealing machine, and place it in an ordinary PCR machine for PCR reaction.

[0065] D. Open the application software of the droplet fluorescence detector, insert the 96-well reaction plate after the PCR reaction directly into the device, detect the PCR reaction of the droplet in each PCR reaction tube, and finally calculate the copy of the gene to be tested according to the Poisson distribution law number.

[0066] Wherein the primer sequences are as follows:

...

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Abstract

The invention discloses a primer, a kit and a method for detecting Pseudomonas aeruginosa and ExoY in water. The sequences and probes of the primers are respectively: upstream primer P.AF, downstream primer P.AR, and probe P.AP , the upstream primer ExoYF, the downstream primer ExoYR, and the probe ExoYP. The purpose of the present invention is to provide a kind of primer suitable for detecting Pseudomonas aeruginosa and ExoY gene in water in order to overcome the deficiencies in the prior art; Another purpose of the present invention is to provide a kind of primer and composition and proportioning Reasonable, easy to use, fast and accurate detection, suitable for dual ddPCR detection kits for Pseudomonas aeruginosa and ExoY genes in water; another purpose of the present invention is to provide a method for detecting Pseudomonas aeruginosa and ExoY genes in water using the above kit Gene method, the method is simple and fast, and the detection result is accurate.

Description

technical field [0001] The invention relates to a primer for detecting Pseudomonas aeruginosa and ExoY in water, and also relates to a double droplet digital PCR detection kit for detecting Pseudomonas aeruginosa and its main effector protein ExoY in water. It also relates to a method for detecting Pseudomonas aeruginosa and ExoY in drinking water by using the kit. Background technique [0002] Pseudomonas aeruginosa Pseudomonas aeruginosa formerly known as Pseudomonas aeruginosa. It is widely distributed in nature and is one of the most common bacteria in soil. All kinds of water, air, skin, respiratory tract and intestinal tract of normal people have this bacteria. The important condition for the existence of this bacterium is a humid environment. The bacterium is a common environmental microorganism. Because it has low nutritional requirements and is good at using various carbon sources and ammoniated compounds as nitrogen sources, it widely exists in water, soil, food...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/06C12N15/11C12R1/385
CPCC12Q1/689C12Q1/686C12Q2600/158C12Q2563/159C12Q2537/143
Inventor 蔡先全丁宁刘恭源李蓉
Owner INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE
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