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Deoxyribozyme probe for detecting staphylococcus aureus as well as application thereof

A deoxyribozyme probe and Staphylococcus technology, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve severe drug resistance, high mortality rate of infectious diseases, difficult treatment, etc. question

Inactive Publication Date: 2018-06-29
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Staphylococcus aureus (Staphylococcus aureus, SA) is a major bacterium that causes food contamination and bacterial food poisoning (the infection caused by Staphylococcus aureus occupies the second place, second only to E. One of the most common pathogenic bacteria that can cause pneumonia and meningitis, the infection caused by it has a high mortality rate and serious drug resistance, which brings great difficulties to the treatment
Food poisoning caused by Staphylococcus aureus is also extremely common, and cases are reported every year, causing great economic losses

Method used

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  • Deoxyribozyme probe for detecting staphylococcus aureus as well as application thereof
  • Deoxyribozyme probe for detecting staphylococcus aureus as well as application thereof
  • Deoxyribozyme probe for detecting staphylococcus aureus as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the synthesis of deoxyribozyme probe (RFD-SA)

[0043] 1. Synthesis of DNAzyme probe (RFD-SA)

[0044] 将ACTCTTCCTAGCFRQGGTTCGATCAAGA(大连宝生物公司)和CACGGATCCTGACAAGGGCCAAGTTAGAATTCTAGCGTAGGGAGAGTCTCCGCCTGCAGCTCCGTCCG利用DNA连接酶进行连接反应,在37℃下反应30分钟,得到具有如下结构的脱氧核酶探针(RFD-SA):ACTCTTCCTAGCFRQGGTTCGATCAAGACACGGATCCTGACAAGGGCCAAGTTAGAATTCTAGCGTAGGGAGAGTCTCCGCCTGCAGCTCCGTCCG,其中,FRQ的结构如 Formula I shows: F is a T base modified by a fluorescent group Fluorescein; Q is a T base modified by a quenching group DABCYL; R represents a single inserted RNA base A. The probe of the present invention is combined with the specific protein in Staphylococcus aureus, and the secondary structure of the probe will be changed after the combination, thereby activating the catalytic activity of the probe, and self-shearing a single RNA molecule in the deoxyribozyme probe After the probe is self-cleaved, a fragment with a size of 81bp can be obtained.

[0045]

[0046] 2. Mix the 5 mL concentra...

Embodiment 2

[0047] Embodiment 2, utilize dPAGE glue to detect the specificity of deoxyribozyme probe (RFD-SA)

[0048] 1. Preparation of detection solution

[0049] Add 20uL of 2x reaction buffer solution and 2uL of deoxyribozyme probe (RFD-SA) aqueous solution into the 200uL reaction tube to obtain the detection solution. The concentration of DNAzyme probe (RFD-SA) in the detection solution was 0.182uM.

[0050] 2x reaction buffer solution is NaCl, MgCl 2 , Tween 20 and 100mM HEPES (pH 9.0) (Sangon Bioengineering (Shanghai) Co., Ltd., product catalog number A100511) mixed the buffer obtained; wherein, the concentration of each solute in the buffer is: 400mM NaCl, 10mM MgCl 2 , the mass fraction is 0.02% Tween 20.

[0051] 2. Preparation of the solution to be tested

[0052] Listeria monocytogenes, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli, Salmonella, Clostridium difficile, Pseudomonas aeruginosa, Shigella, and Vibrio parahaemolyticus were cultured in LB medium...

Embodiment 3

[0060] Embodiment 3, detect the sensitivity of deoxyribozyme probe (RFD-SA) on microplate reader

[0061] 1. Add 2uL deoxyribozyme probe (RFD-SA) aqueous solution with a concentration of 2uM and 40uL 2x reaction buffer in each well of a 96-well plate, and deoxyribozyme probe (RFD-SA) in the detection solution The concentration in is 0.048uM. Set up parallel, controlled experiments.

[0062] 2. Add 40uL of Staphylococcus aureus containing different volumes (0.008uL, 0.02uL, 0.04uL, 0.08uL, 0.2uL, 0.4uL, 0.8uL, 2uL, 4uL, 8uL) into the wells with an optical density value of 0.9 The LB culture fluid of the cell culture supernatant was obtained to obtain the LB culture fluid containing different concentrations of the Staphylococcus aureus culture supernatant; and the LB culture fluid was used as a negative control.

[0063] Add 40uL containing different volumes (0.008uL, 0.02uL, 0.04uL, 0.08uL, 0.2uL, 0.4uL, 0.8uL, 2uL, 4uL, 8uL) of Staphylococcus aureus cell culture with an opti...

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Abstract

The invention discloses a deoxyribozyme probe for detecting staphylococcus aureus as well as application thereof. The structure of the deoxyribozyme probe for detecting staphylococcus aureus, which isprovided by the invention, is as follows: ACTCTTCCTAGCFRQGGTTCGATCAAGACACGGATCCTGACAAGGGCCAAGTTAGAATTCTAGCGTAGGGAGAGTCTCCGCCTGCAGCTCCGTCCG, wherein F is T base modified by a fluorescence group; Q isT base modified by a quenching group; and R represents singly inserted RNA base A. An experiment proves that the probe can perform rapid, accurate and high-sensitivity detection on the staphylococcusaureus.

Description

technical field [0001] The invention relates to a deoxyribozyme probe for detecting Staphylococcus aureus and its application, belonging to the field of biotechnology. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, SA) is a major bacterium that causes food contamination and bacterial food poisoning (the infection caused by Staphylococcus aureus occupies the second place, second only to E. One of the most common pathogenic bacteria can cause pneumonia and meningitis. The infection caused by it has a high mortality rate and serious drug resistance, which brings great difficulties to the treatment. Food poisoning caused by Staphylococcus aureus is also very common, and there are case reports every year, causing great economic losses. [0003] At present, all countries in the world have listed Staphylococcus aureus as a statutory inspection item for food hygiene. Early diagnosis and treatment are the key to improving the cure rate of Staphylococcus...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/14C12N15/11C12R1/445
CPCC12Q1/689
Inventor 蒋宇扬谭英曲龙何盛南谭春燕刘峰
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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