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Molecular beacon probes and kit for rapidly assaying ethambutol-resistant mycobacterium tuberculosis

A technology of molecular beacon probes and Mycobacterium tuberculosis, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of false negatives, long detection cycle, and high detection cost. Achieve the effects of high specificity, rapid detection, and good fluorescence signal intensity

Inactive Publication Date: 2016-07-20
SUZHOU ZHONGSHENG DAMAIDI MOLECULE DIAGNOSTICS TECH
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Problems solved by technology

Among them, the bacterial culture method is the gold standard for detecting ethambutol-resistant Mycobacterium tuberculosis, but this method requires drug susceptibility testing, usually takes 15-20 days to see the final test results, and the cost of testing is high and the testing cycle is long ;Using the PCR method can increase the geometric progression of the copy number of a specific nucleic acid sequence in a short period of time, which has high sensitivity and specificity, but the disadvantage is that the false positive rate is high. On the other hand, samples often contain substances that inhibit the PCR reaction. Components, which will lead to the emergence of false negatives, the shortcomings of these two aspects limit the clinical application of PCR detection

Method used

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  • Molecular beacon probes and kit for rapidly assaying ethambutol-resistant mycobacterium tuberculosis
  • Molecular beacon probes and kit for rapidly assaying ethambutol-resistant mycobacterium tuberculosis

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Embodiment 1

[0031] Example 1: Design and synthesis of molecular beacon probes and oligonucleotide sequences

[0032] Select the embABC operon gene sequences that can specifically detect ethambutol-resistant Mycobacterium tuberculosis, and design molecular beacon probes that are completely complementary to these gene sequences based on thermodynamic characteristics and higher-level structures:

[0033] BeaconEMB1:

[0034] 5'-Cy3-ATCGTCTTGCGCCCATTGTGCAATAACGAT-BHQ1-3';

[0035] BeaconEMB2:

[0036] 5'Cy3-CACTGGTCATGCAACATCTACTCCAGTG-BHQ1-3';

[0037] BeaconEMB3:

[0038] 5'-Cy3-CACTGCTAAACCCCGGAAAGGGTCTCAGTG-BHQ1-3'.

[0039] The 5' end of the probe is labeled with CY3, the 3' end is labeled with BHQ1, and the excitation wavelength of the fluorophore is 552nm.

[0040] The ten bases at the end of the probe are complementary (the ten bases on the same chain in bold above), so that the probe can form a neck loop structure and will not form a higher-order structure with the probe itself. ...

Embodiment 2

[0041] Embodiment 2: The comparative experiment of three kinds of methods detection sputum sample

[0042] 1. Roche culture method

[0043] The modified Lowenstein-Jensen medium was used to culture the treated sputum. Specifically, add 2 mL of 4% sodium hydroxide to 1 mL of sputum sample, vortex and oscillate for 30 seconds to mix, leave at room temperature for 20 minutes, and oscillate 2 to 3 times during the process to promote liquefaction of sputum. Take 0.1 mL of digested sputum with a sterile graduated capillary pipette, inoculate in parallel on the non-resistant and ethambutol-resistant medium slant, and culture in a 37°C incubator. The culture was observed on the 3rd and 7th day after inoculation, and then observed once a week until the end of the 8th day. If the culture is positive, report it at any time. If no bacterial growth is found after 8 weeks of culture, the report is culture negative.

[0044] 2. PCR detection

[0045] Bacterial DNA in the samples was extr...

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Abstract

The invention provides molecular beacon probes for rapidly assaying ethambutol-resistant mycobacterium tuberculosis. The molecular beacon probes include Beacon EMB1, Beacon EMB2 and Beacon EMB3. By means of the signal synergy of the three molecular beacon probes, with the embABC gene of mycobacterium tuberculosis as a target spot, ethambutol-resistant mycobacterium tuberculosis is assayed. The probes in the invention have high fluorescence intensity and high specificity, and can effectively and rapidly assay ethambutol-resistant mycobacterium tuberculosis. The invention also discloses a kit and an assay method for rapidly assaying ethambutol-resistant mycobacterium tuberculosis.

Description

technical field [0001] The invention relates to a molecular beacon probe, in particular to a molecular beacon probe and a kit for rapidly detecting ethambutol-resistant Mycobacterium tuberculosis. 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. The occurrence and prevalence of drug-resistant tuberculosis is a difficult problem and the biggest challenge for tuberculosis control at present and in the future. With the development of molecular biology techniques and the gradual elucidation of the mechanism of tuberculosis drug resistance at the gene level, the establishment of molecular biology detection methods for drug resistance can increase the speed of detection. Screening drug-resistant tuberculosis patients as soon as possible...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6841C12Q1/04C12Q2563/107
Inventor 方国伟洪冉
Owner SUZHOU ZHONGSHENG DAMAIDI MOLECULE DIAGNOSTICS TECH
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