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CPA detection primer, kit and method for pseudomonas aeruginosa

A Pseudomonas aeruginosa detection kit technology, applied in the biological field, can solve the problem of high difficulty in designing reaction primers, reduce the probability of false positive results, ensure reliability, and improve the effect of disease diagnosis

Pending Publication Date: 2020-03-13
SOUTH CHINA UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the design of primers for this reaction is very difficult, and it is necessary to design five primers in a limited product length, and to avoid non-specific amplification of the five primers themselves and affect the results, so the design of primers is particularly important

Method used

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  • CPA detection primer, kit and method for pseudomonas aeruginosa
  • CPA detection primer, kit and method for pseudomonas aeruginosa
  • CPA detection primer, kit and method for pseudomonas aeruginosa

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] 1. Design primers

[0052] According to the amplification reaction principle of cross-primed constant temperature amplification (CPA), the following primers were designed for the target oprL using Primer Premier software, including stripping primers 4s and 5a, cross-amplification primers 2a1s, and specific primers 2a and 3a; The nucleotide sequences are as follows:

[0053] Target oprL stripping primer 4s: 5'-GACCCGAACGCAGGCTAT-3' (SEQ ID NO.1);

[0054] Target oprL stripping primer 5a: 5'-CGCTGCCTTTCAGGTCTTT-3' (SEQ ID NO.2);

[0055] Target oprL cross primer 2a1s: 5'-GCATGGCTTCCGGCTTCA GTGCGATCACCACCTTCTACTT-3' (SEQ ID NO.3);

[0056] Target oprL specific primer 2a: 5'-GCATGGCTTCCGGCTTCA-3' (SEQ ID NO.4);

[0057] Target oprL specific primer 3a: 5'-GTCGGAGCTGTCGTACTC-3' (SEQ ID NO.5).

Embodiment 2

[0059] 1. The method for detecting pathogenic microorganisms based on cross-primer constant temperature amplification reaction technology. In this embodiment, Pseudomonas aeruginosa is used as an example. The reagents used are as follows:

[0060] a. The stripping primers 4s and 5a, the cross-amplification primers 2a1s, and the specific primers 2a and 3a with a concentration of 10 μM, the primer sequences are as shown in SEQ ID NO.1-5 in Example 1;

[0061] b. Prepare 2× reaction stock solution:

[0062] ①20×reaction stock solution: containing 0.2M potassium chloride, 0.2M ammonium sulfate, 40mM~240mM magnesium sulfate and 2% (v / v) Tween 20 (both final concentrations, prepared with nucleic acid-free water); The concentration of magnesium sulfate set in the embodiment is 40mM, 80mM, 120mM, 160mM, 200mM, 240mM;

[0063] ②2× reaction stock solution: 100 μL 20× reaction stock solution, 500 μL 3.2M betaine, 280 μL 10mM dNTPs, 27 μL 1.5M Tris-HCl, 93 μL nucleic acid-free water;

...

Embodiment 3

[0073] The method for detecting Pseudomonas aeruginosa based on cross-primer constant temperature amplification reaction technology comprises the following steps:

[0074]1. The method for detecting pathogenic microorganisms based on cross-primer constant temperature amplification reaction technology. In this embodiment, Pseudomonas aeruginosa is used as an example. The reagents used are as follows:

[0075] a. The stripping primers 4s and 5a, the cross-amplification primers 2a1s, and the specific primers 2a and 3a with a concentration of 10 μM, the primer sequences are as shown in SEQ ID NO.1-5 in Example 1;

[0076] b. Prepare 2× reaction stock solution:

[0077] ① 20× reaction stock solution: containing 0.2M potassium chloride, 0.2M ammonium sulfate, 160mM magnesium sulfate and 2% (v / v) Tween 20 (both final concentrations, prepared with nucleic acid-free water);

[0078] ②2× reaction stock solution: 100 μL 20× reaction stock solution, 500 μL 3.2M betaine, 280 μL 10mM dNTPs...

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Abstract

The invention discloses a CPA detection primer, a kit and a method for pseudomonas aeruginosa. The CPA detection primer is designed for a target oprL, and has nucleotide sequences as shown in SEQ ID NO. 1-5. The invention also provides a CPA detection kit for the pseudomonas aeruginosa. The CPA detection kit has the advantages of high sensitivity, good specificity, good applicability, simple, convenient and rapid operation, accurate and reliable result, low detection cost and the like. The CPA detection primer or the CPA detection kit is utilized for cross a primer isothermal amplification reaction on a to-be-detected sample; results can be directly judged and read by naked eyes through fluorescent dye color development; and whether the to-be-detected sample contains the pseudomonas aeruginosa or not can be quickly and accurately detected, which has important significance for improving the disease diagnosis rate of important groups and diagnosing diseases in an early stage.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a CPA detection primer, kit and method for Pseudomonas aeruginosa. Background technique [0002] At present, the detection and identification methods of microorganisms are mainly divided into culture identification, immunoassay and nucleic acid detection. The traditional culture identification method is cumbersome to operate and the experiment period is long. The immunoassay method has high requirements for experimenters, and the nucleic acid detection mainly focuses on PCR detection, which takes a long time and has low sensitivity. [0003] Compared with other nucleic acid amplification technologies, crossing primer constant temperature amplification (Crossing Priming Amplification, CPA) technology can quickly, efficiently and specifically amplify target sequences under isothermal conditions, and is easy to operate, does not require precise temperature-changing equipment, and costs ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/385
CPCC12Q1/689C12Q1/6844C12Q2527/125C12Q2527/101
Inventor 徐振波骆玉婷陈玲刘君彦梁毅毛雨竹陈雁妮石帆彭瑞欣陈锦璇
Owner SOUTH CHINA UNIV OF TECH
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