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CPA primer for killing leukotoxin positive staphylococcus aureus, kit and detection method

A technology for killing leukotoxin and staphylococcus, which is applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve problems such as insufficient detection sensitivity, achieve long detection cycle improvement, increase diagnostic rate and accuracy The effect of high reliability and low detection cost

Pending Publication Date: 2021-10-19
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem of insufficient detection sensitivity of existing methods, the object of the present invention is to provide a CPA primer for leukotoxin-positive Staphylococcus aureus and its detection kit and detection method

Method used

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  • CPA primer for killing leukotoxin positive staphylococcus aureus, kit and detection method
  • CPA primer for killing leukotoxin positive staphylococcus aureus, kit and detection method
  • CPA primer for killing leukotoxin positive staphylococcus aureus, kit and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0041] The method for detecting leukocidal toxin-positive Staphylococcus aureus based on cross-primer constant temperature amplification technology comprises the following steps:

[0042]1. In this embodiment, leukocidal toxin-positive Staphylococcus aureus is taken as an example, and the reagents used are as follows:

[0043] a. The stripping primers 4s and 5a, the cross-amplification primers 2a1s, and the specific primers 2a and 3a each at a concentration of 10 μM, the primer sequences are as shown in the preceding SEQ ID NO.1-SEQ ID NO.5;

[0044] b.2×Reaction stock solution: Tris-HCl with concentration of 40.0mM, ammonium sulfate of 20.0mM, potassium chloride of 20.0mM, magnesium sulfate of 16.0mM, Tween 20 of 0.2% (v / v), 1.4M Betaine, 10.0mM dNTPs (each) composition;

[0045] c. Bst DNA polymerase (large fragment, NEB company) aqueous solution with a concentration of 8U / μL;

[0046] d. Mixed solution of calcein and manganese chloride: first prepare a calcein solution wi...

Embodiment 2

[0059] The cross constant temperature amplification reaction detection aureus leukocidal toxin primer screening test comprises the following steps:

[0060] The detection primers Pvl-1 and Pvl-2 were established according to the reaction system and conditions in Example 1 to establish a detection method for the cross thermostatic amplification reaction, and the primer screening test was carried out.

[0061] Design the corresponding CPA detection primers for the target pvl. The primer screening process is to use the designed 4s and 5a primers as the upstream and downstream primers of the PCR reaction to analyze whether the target region can be amplified using the corresponding stripping primers, and at the same time analyze the stripping primers. have high specificity. When a single band appears in the reaction and corresponds to the size of the product, it indicates that the primer has good specificity. If non-specific amplification of the stripped primer occurs, a ladder-lik...

Embodiment 3

[0078] Cross constant temperature amplification reaction detection aureus leukocidal toxin specificity test, comprising the following steps:

[0079] Genomic DNA of leukocidal toxin-positive Staphylococcus aureus and non-Staphylococcus aureus was used to establish a cross-isothermal amplification reaction detection method according to the reaction system and conditions in Example 1, and a specificity test was performed.

[0080] Among them, non-staphylococcus aureus are: Pseudomonas aeruginosa ATCC27853; Pseudomonas aeruginosa ATCC1014; Pseudomonas aeruginosa ATCC15442; Pseudomonas aeruginosa ATCC17934; Special bacteria ATCC19115; Listeria monocytogenes ATCC19114; Escherichia coli ATCC43895; Escherichia coli E019; Escherichia coli E020; Escherichia coli E043; Escherichia coli E044; Vibrio parahaemolyticus ATCC17802; .

[0081] Escherichia coli E019, Escherichia coli E020, Escherichia coli E043 and Escherichia coli E044 involved in the present invention have been published in ...

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Abstract

The invention discloses a CPA primer for killing leukotoxin positive staphylococcus aureus, a detection kit and a detection method of the CPA primer. The CPA primer designed aiming at a target spot pvl comprises stripping primers 4s and 5a, a cross amplification primer 2a1s and specific primers 2a and 3a; the nucleotide sequences of the primers are respectively as shown in SEQ ID NO.1 to SEQ ID NO.5. According to the invention, a pair of stripping primers, cross primers and specific primers are designed aiming at a specific target sequence pvl of the leukotoxin-killing staphylococcus aureus, a cross primer isothermal amplification reaction system is constructed, the staphylococcus aureus carrying the leukotoxin-killing gene pvl can be detected in about 60 minutes, and the detection limit reaches the level of pg / [mu]L; the defects of long period, low sensitivity, high cost, difficulty in field application and the like of the existing detection technology are overcome.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a CPA primer for leukocidal toxin-positive Staphylococcus aureus, a detection kit and a detection method thereof. Background technique [0002] Staphylococcus aureus is one of the common clinical opportunistic pathogens, which often reside in the nasal cavity, throat, skin and mucous membranes of humans and animals. Staphylococcus aureus can produce various aggressive enzymes and toxins, such as leukocidal toxin, plasma coagulase, hemolytic toxin, toxic shock syndrome toxin, enterotoxin, etc. Under certain conditions, Staphylococcus aureus can cause or aggravate a series of inflammatory infections on the human body through the comprehensive regulation of various virulence factors and invasion enzymes, causing boils, folliculitis, bacteremia, bacteremia, necrotic diseases such as pneumonia. [0003] As one of the important virulence factors of Staphylococcus aureu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2537/1376C12Q2563/107Y02A50/30
Inventor 徐振波刘子奇骆玉婷刘君彦陈玲叶燕锐李晓玺洪玮彭芳付欣户帅锋苏健裕
Owner SOUTH CHINA UNIV OF TECH
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