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Aptamer based methicillin-resistant staphylococcus aureus fluorescence detection method

A technology for methicillin-resistant Staphylococcus aureus, applied in the field of fluorescence detection of methicillin-resistant Staphylococcus aureus based on aptamers, can solve the problems of cumbersome preparation process, high price and high cost of monoclonal antibodies, and achieve easy preparation and labeling, increased sensitivity, and reduced cost

Active Publication Date: 2018-12-11
WEIFANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the ELISA method and the latex agglutination method shorten the detection period, since the Staphylococcus aureus protein A can bind to the Fc fragment of the mammalian IgG antibody, in order to specifically detect PBP2a, the PBP2a protein must be extracted from the bacteria, and the reagent The preparation process of the monoclonal antibody used in the box is cumbersome and expensive
At present, latex agglutination kits still rely on imports, which are expensive

Method used

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  • Aptamer based methicillin-resistant staphylococcus aureus fluorescence detection method
  • Aptamer based methicillin-resistant staphylococcus aureus fluorescence detection method
  • Aptamer based methicillin-resistant staphylococcus aureus fluorescence detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] (1) The magnetic nanoparticles used in the examples of the present invention are carboxyl-modified magnetic nanoparticles with a particle size of 800 nm, purchased from Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd. PBP2a aptamer, synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., address: No. 698, Xiangmin Road, Songjiang District, Shanghai, preservation number: M23

[0043] The sequence of the PBP2a aptamer is:

[0044] 5'-CCATCCACACTCCGCAAGGGTGCCCCGGGGGGCTGTTCAGCGTGGTGGTGGGATGCCGTTTTGGTCCTTAGTCTCCGTCGTCGGCTGCCTCTACAT-3', the 5' end is labeled with the fluorescent group FITC;

[0045] The complementary DNA strand sequences of the aptamers are: 5'-GCGGAGTGTGGATGG-DABCYL-3'(Q-DNA-1),

[0046] 5'-CCTTGCGGAGTGTGG-DABCYL-3'(Q-DNA-2) and

[0047] 5'-CACCCTTGCGGAGTG-DABCYL-3'(Q-DNA-3), the 3' end is labeled with the quenching group DABCYL.

[0048] (2) In this example, based on the above-mentioned synthesized fluorescently labeled PBP2a aptamer and que...

Embodiment 2

[0068] (1) The magnetic nanoparticles used in the examples of the present invention are carboxyl-modified magnetic nanoparticles with a particle size of 800 nm, purchased from Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd. PBP2a aptamer, synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., address: No. 698, Xiangmin Road, Songjiang District, Shanghai, preservation number: M25.

[0069] The sequence of the PBP2a aptamer is:

[0070] 5'-CCATCCACACTCCGCAAGGGTCGGTCTTCCCCTTCAGCTTGATGGGGTCCTGGGTGCCGGGATTTGGTTGCTGGTCTTCGTCGGCTGCCTCTACAT-3', the 5' end is labeled with the fluorescent group FITC; the complementary DNA strand sequence of the aptamer is: 5-CCTTGCGGAGTGTGG-DABCYL-3'(Q-DNA-2), the 3' end is labeled with a quenching group DABCYL.

[0071] (2) In this example, based on the above-mentioned synthesized fluorescently labeled PBP2a aptamer and quencher labeled aptamer complementary DNA strand, a highly sensitive and highly selective fluorescent detection syste...

Embodiment 3

[0084] (1) The magnetic nanoparticles used in the examples of the present invention are carboxyl-modified magnetic nanoparticles with a particle size of 800 nm, purchased from Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd. PBP2a aptamer, synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., address: No. 698, Xiangmin Road, Songjiang District, Shanghai, preservation number: M23

[0085] The sequence of the PBP2a aptamer is:

[0086] 5'-CCATCCACACTCCGCAAGGGTGCCCCGGGGGGCTGTTCAGCGTGGTGGTGGGATGCCGTTTTGGTCCTTAGTCTCCGTCGTCGGCTGCCTCTACAT-3', the 5' end is labeled with the fluorescent group FITC;

[0087] The complementary DNA strand sequences of the aptamers are: 5'-GCGGAGTGTGGATGG-DABCYL-3'(Q-DNA-1),

[0088] 5'-CCTTGCGGAGTGTGG-DABCYL-3'(Q-DNA-2) and

[0089] 5'-CACCCTTGCGGAGTG-DABCYL-3'(Q-DNA-3), the 3' end is labeled with the quenching group DABCYL.

[0090] (2) In this example, based on the above-mentioned synthesized fluorescently labeled PBP2a aptamer and que...

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Abstract

The invention discloses an aptamer based methicillin-resistant staphylococcus aureus fluorescence detection method and application thereof. The detection method comprises the following steps of (1) sequentially adding IgG antibody coated magnetic nano-particles and a sample to detected in a vessel, and performing uniform mixing and incubation to enable the antibody coated magnetic nano-particles to be fully combined with staphylococcus aureus in the sample; (2) collecting the magnetic nano-particles with an applied magnetic field, performing washing, adding a staphylococcus aureus cracking reagent, performing incubation for a period of time, and collecting a bacterium lysate under the effect of the applied magnetic field; (3) adding the bacterium lysate in a complementary DNA chain hybridof a fluorescence labeled PBP2a protein aptamer and a quencher group labeled aptamer, and performing uniform mixing and incubation; and (4) determining fluorescence changes of the aptamers and the complementary DNA chain hybrid thereof before and after addition of the bacterium lysate, judging whether PBP2a exists or not according to fluorescence change values, and realizing rapid detection of methicillin-resistant staphylococcus aureus in a clinic sample.

Description

technical field [0001] The invention relates to the field of drug-resistant bacteria detection, in particular to an aptamer-based fluorescence detection method and application of methicillin-resistant Staphylococcus aureus. Background technique [0002] Staphylococcus aureus is one of the most common pathogens of nosocomial infection, which can cause serious diseases such as skin and soft tissue infection, bacteremia, endocarditis, pneumonia, bone and joint infection, and central nervous system infection. The irrational use of antibiotics in recent years has led to an increasing number of methicillin-resistant Staphylococcus aureus (MRSA). Due to the rapid spread and wide prevalence of MRSA, except for methicillin resistance, it is also resistant to other β-lactam and cephalosporin antibiotics, and it is resistant to aminoglycosides, macrolides, tetracyclines, and fluoroquinolones. Nullones, sulfonamides, and rifampicin also produce varying degrees of drug resistance, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56938
Inventor 乔晋娟伊正君孟祥英李恒
Owner WEIFANG MEDICAL UNIV
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