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CPA primers and kit for detection of methicillin-resistant staphylococcus aureus, and detection method

A methicillin-resistant Staphylococcus aureus and methicillin-resistant technology, applied in the field of biotechnology detection, can solve problems such as insufficient sensitivity, limited scope of application, etc., and achieve the effects of simple and fast operation, good applicability, and shortened cycle

Active Publication Date: 2019-09-03
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection limit of this patent only reaches the pg / μL level, and the sensitivity is not high enough, which limits the scope of application

Method used

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  • CPA primers and kit for detection of methicillin-resistant staphylococcus aureus, and detection method
  • CPA primers and kit for detection of methicillin-resistant staphylococcus aureus, and detection method
  • CPA primers and kit for detection of methicillin-resistant staphylococcus aureus, and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The method for detecting methicillin-resistant Staphylococcus aureus based on cross-primer constant temperature amplification (CPA) reaction technology may further comprise the steps:

[0046] (1) Reagents used:

[0047] 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.10;

[0048] 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;

[0049] c. Bst DNA polymerase (large fragment, NEB company) aqueous solution with a concentration of 8U / μL;

[0050] d. Mixed solution of calcein and manganese chloride: first prepare a calcein solution with a concentration of 50 μM (dissolved in dimethyl sulfoxide); then take 25 μL of 50 μM calcein solu...

Embodiment 2

[0060] Cross constant temperature amplification reaction (CPA) detection methicillin-resistant Staphylococcus aureus specific test, comprises the following steps:

[0061] The genomic DNA of methicillin-resistant Staphylococcus aureus NCTC10442 and non-methicillin-resistant Staphylococcus aureus was established according to the reaction system and conditions in Example 1 to establish a cross-isothermal amplification reaction detection method, and a specificity test was carried out;

[0062] Among them, the non-methicillin-resistant Staphylococcus aureus is: Escherichia coli O157:H7E019; Escherichia coli O157:H7E020; Escherichia coli E043; Escherichia coli E044; Listeria ATCC19116; Listeria monocytogenes ATCC19114; Listeria monocytogenes ATCC19115; Listeria monocytogenes ATCC15313; Listeria monocytogenes ATCC19113; Pseudomonas aeruginosa ATCC27853; Vibrio parahaemolyticus ATCC17802; ATCC27969; Lactobacillus casei BM-LC14617.

[0063] The genome of methicillin-resistant Staphyl...

Embodiment 3

[0065] Cross constant temperature amplification reaction (CPA) detects the sensitivity comparison test of methicillin-resistant Staphylococcus aureus, comprising the following steps:

[0066] The genome of methicillin-resistant Staphylococcus aureus NCTC10442 was serially diluted 10 times to 3.0ng / μL, 300pg / μL, 30pg / μL, 3pg / μL, 300fg / μL, 30fg / μL, 3fg / μL, 300ag / μ L, set negative control (nucleic acid-free water) simultaneously, construct the cross constant temperature amplification method according to the reaction system among the embodiment 1 and carry out 2% agarose gel electrophoresis to the amplified product, to determine the sensitivity of detection method.

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Abstract

The invention discloses CPA primers and a detection kit for detection of methicillin-resistant staphylococcus aureus, and a detection method. The CPA primers are designed for two targets femA and mecAand comprise a stripping primer 4s and 5a, a cross-amplification primer 2a1s, and a specific primer 2a and 3a, wherein the primers have the sequences shown in SEQ ID NO.1-SEQ ID NO.10. The detectionmethod comprises the steps: establishing a cross-primer constant temperature amplification reaction system for detecting femA and mecA, carrying out cross-primer constant temperature amplification reaction, observing the color changes of the two reaction systems, and if colors of both the reaction systems turn into green, indicating that a sample to be tested contains methicillin-resistant staphylococcus aureus; otherwise, indicating that the sample to be tested does not contain methicillin-resistant staphylococcus aureus. The detection time of the primers and method is fast, and the detectionresult can be obtained in about 60 min. In addition, the detection sensitivity is high and reaches the level of f g / [mu]L.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a CPA primer for detecting methicillin-resistant staphylococcus aureus, a detection kit and a detection method thereof. Background technique [0002] At present, the detection and identification methods of microorganisms are mainly divided into culture identification, immunoassay and nucleic acid detection. The culture identification method and immunoassay method are cumbersome to operate, the experiment cycle is long, and the professional level of the experimenters is high. [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 It shows broad development prospects...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/14C12Q1/6844C12N15/11C12R1/445
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107Y02A50/30
Inventor 徐振波骆玉婷刘君彦陈玲苏健裕李冰李琳李晓玺张霞
Owner SOUTH CHINA UNIV OF TECH
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