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Molecular beacon probe for quickly detecting non-Mycobacterium tuberculosis and detection method using same

A technology of molecular beacon probes and mycobacteria, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and measurement/testing of microorganisms, can solve the problem of inability to distinguish tuberculosis mycobacteria from non-tuberculous mycobacteria, Can not get positive results, low sensitivity and other problems, to achieve the effect of rapid detection, high specificity, high signal intensity

Active Publication Date: 2014-09-10
SUZHOU ZHONGSHENG DAMAIDI MOLECULE DIAGNOSTICS TECH
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Problems solved by technology

[0003] At present, the most commonly used method for clinical detection of Mycobacterium tuberculosis and Non-tuberculosis Mycobacteria is acid-fast staining microscopy. This method has the advantages of simplicity, rapidity, and low cost. Positive results are often not obtained when the amount of bacteria is small, and it is impossible to distinguish between Mycobacterium tuberculosis and Mycobacterium nontuberculous

Method used

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  • Molecular beacon probe for quickly detecting non-Mycobacterium tuberculosis and detection method using same

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Embodiment 1

[0037] Example 1: Design and synthesis of molecular beacon probes and oligonucleotide sequences

[0038] Select a target sequence that can specifically detect non-tuberculous mycobacteria, and design a molecular beacon probe that is completely complementary to the target sequence:

[0039] Beacon NTM: (5'-CY3-CATTGTACGCCCAGTAATTCCGGACCAATG–BHQ1-3');

[0040] The molecular beacon is a specific sequence of a neck ring structure composed of bases. The 5' end of the probe is labeled with Cy3, and the 3' end is labeled with BHQ1. The excitation wavelength of the fluorescent group is 552nm, and the detection wavelength is 570nm; artificially designed synthetic molecules The beacon and its fully complementary oligonucleotide (5'-GTCCGGAATTACTGGGCGTA-3'). Through thermal denaturation curve experiments on molecular beacons and oligonucleotides, it was determined that the optimal reaction temperature of fluorescence in situ hybridization was 52°C, and the optimal concentration of deion...

Embodiment 2

[0041] Example 2: Detection of mycobacterial standard strains using Mycobeacon Probe FISH.

[0042] Mycobacterium standard strains were purchased from ATCC, and a total of 5 tuberculosis mycobacteria and 12 nontuberculous mycobacteria were detected.

[0043] The test kit includes:

[0044] (1) Lysis solution: 4% (w / v) sodium hydroxide;

[0045](2) Hybridization solution: 10% (w / v) dextran sulfate, 10mM NaCl, 20% (v / v) formamide, 0.1% (w / v) sodium pyrophosphate, 0.2% (w / v) poly Vinylpyrrolidone, 0.2% (w / v) polysucrose, 5mM Na2EDTA, 0.1% (v / v) TritonX-100, 50mM Tris / HCl (pH7.5), 10ng / μL molecular beacon probe, the The base sequence of the molecular beacon probe is:

[0046] Beacon NTM: 5'-CY3-CATTGTACGCCCAGTAATTCCGGACCAATG–BHQ1-3';

[0047] (3) Termination solution: 1% (v / v) methyl disulfuric acid;

[0048] (4) Washing solution: 5mM Tris, 15mM NaCl, 0.1% (v / v) Triton X-100, pH 10.

[0049] Detection method:

[0050] (1) Take 10 μL of sputum sample and drop it on a glass s...

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Abstract

The invention discloses a molecular beacon probe for quickly detecting non-Mycobacterium tuberculosis. The invention is characterized in that the base sequence of the molecular beacon probe is BeaconNTM: 5'-CY3-(b)CATTG( / b)TACGCCCATAATTCGGAC(b)CAATG( / b)-BHQ1-3', wherein the 5' terminal of the probe is marked with Cy3, the 3' terminal is marked with BHQ1, the fluorophore excitation wavelength is 552nm, and the detection wavelength is 570nm. The molecular beacon probe has the advantages of high signal intensity and high specificity, and can effectively and quickly detect non-Mycobacterium tuberculosis. The invention also discloses a kit and detection method for quickly detecting non-Mycobacterium tuberculosis.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a molecular beacon probe and a kit for detecting a small amount of non-tuberculous mycobacteria in a sample by means of fluorescence in situ hybridization. Background technique [0002] According to the results of the Fourth National Tuberculosis Epidemic Sampling Survey (2000) organized by the Ministry of Health of China, 400 million people in my country have been infected with Mycobacterium tuberculosis. It ranks second in the world, after India. According to the National Infectious Disease Epidemic Report released by the Chinese Ministry of Health in March 2006, tuberculosis still ranks first in the number of cases and deaths among Class A and Class B infectious diseases. Most of the tuberculosis patients or residents living in areas with high incidence of tuberculosis are difficult to get a rapid and accurate diagnosis. Although the characteristics of non-tubercu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/32
CPCC12Q1/6841C12Q2563/107C12Q2525/301
Inventor 洪冉方华成刘振世
Owner SUZHOU ZHONGSHENG DAMAIDI MOLECULE DIAGNOSTICS TECH
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