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Primer probe combination for identifying mycobacterium abscessus and mycobacterium massiliense

A primer probe, mycobacteria technology, applied in the direction of microorganism-based methods, microorganisms, recombinant DNA technology, etc., can solve the problem that the cure rate is only about 60%

Inactive Publication Date: 2018-11-23
BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Mycobacterium abscessus complex is naturally resistant to first-line anti-tuberculosis drugs, and most of them are ineffective against common antibacterial drugs. The treatment plan is usually a multi-drug combination plan with clarithromycin and other macrolide antibiotics as the core, and the cure rate is only About 60%, so NTM disease is the focus and difficulty of treatment

Method used

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  • Primer probe combination for identifying mycobacterium abscessus and mycobacterium massiliense
  • Primer probe combination for identifying mycobacterium abscessus and mycobacterium massiliense
  • Primer probe combination for identifying mycobacterium abscessus and mycobacterium massiliense

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, the design of primer probe combination

[0054] A large number of sequence analyzes and comparisons of Mycobacterium abscessus and Mycobacterium marseille were carried out, and the influencing factors of clarithromycin drug susceptibility of Mycobacterium abscessus and Mycobacterium marseille were analyzed to obtain a set of Primer-probe combinations for Bacillus and M. marseilles are as follows:

[0055] Primer F1 (sequence 1 of the sequence listing): 5'-GCGACGCCAGTGGGGCTGGT-3';

[0056] Primer R1 (sequence 2 of the sequence listing): 5'-CGCCGCCTGATCACCAGCAC-3';

[0057]Probe P1 (SEQ ID NO: 3 of the Sequence Listing): 5'-GCGGATCGTCGCCGAATCCGGT-3'

[0058] Probe P2 (Sequence 4 of the Sequence Listing) 5'-CGCCCTACCAAGTCACCAGCG-3'.

[0059] The 5' end of probe P1 was modified with FAM fluorescent group, and the 3' end was modified with BHQ1 quencher group.

[0060] The 5' end of probe P2 was modified with HEX fluorescent group, and the 3' end was modifie...

Embodiment 2

[0062] Embodiment 2, establishment of detection method

[0063] 1. Extract the total DNA of the sample to be tested.

[0064] 2. Using the total DNA obtained in step 1 as a template, perform fluorescent quantitative PCR detection.

[0065] Fluorescence quantitative PCR reaction system: primer F1, primer R1, probe P1, probe P2 each 1 μl, PCR buffer + dNTPs + Mg 2+ (PCR mix) 25μl, DNA template 4μl, RNase Free H 2 O 12 μl.

[0066] The concentration of each primer in the system is 500nM, and the concentration of each probe in the system is 250nM.

[0067] DNA was added to the reaction system in the form of DNA solution, and the concentration of DNA in the DNA solution was 10 ng / μl.

[0068] Fluorescent quantitative PCR reaction program: pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 5 seconds, annealing at 58°C for 20 seconds, extension at 72°C for 20 seconds, a total of 40 cycles.

[0069] At the same time, a positive control group and a negative control g...

Embodiment 3

[0079] Embodiment 3, standard bacterial strain detection

[0080] Strains to be tested: Mycobacterium abscessus (ATCC19977), Mycobacterium avium (ATCC25291), Mycobacterium intracellulare (ATCC13950), Mycobacterium kansasii (ATCC12478) and Mycobacterium marseille standard strain (CIP108297, purchased from Paris, France Stuart Institute).

[0081] The method in Example 2 is adopted to detect, and the results show that when the sample to be tested is Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium kansasii, the FAM signal is negative, and the HEX signal is negative simultaneously; when the sample to be tested is For Mycobacterium abscessus, the FAM signal is positive and the HEX signal is positive; when the sample to be tested is the standard strain of Mycobacterium marseille, the FAM signal is positive and the HEX signal is negative.

[0082] The above method has good specificity.

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Abstract

The invention discloses a primer probe combination for identifying mycobacterium abscessus and mycobacterium massiliense. The primer probe combination provided by the invention consists of a single chain DNA shown as sequences 1 to 4 in a sequence table. The invention provides an identification method of mycobacterium abscessus and mycobacterium massiliense; the distinguishing on the mycobacteriumabscessus and the mycobacterium massiliense can be fast completed in the molecular level; the characteristics of simplicity, convenience, high speed, high specificity and high sensitivity are realized, so that the problem that the mycobacterium abscessus composite groups cannot be accurately and fast identified by the existing molecular technology is solved.

Description

technical field [0001] The invention relates to a combination of primers and probes for identifying Mycobacterium abscessus and Mycobacterium marseilles. Background technique [0002] Nontuberculous mycobacteria (NTM) refers to all mycobacteria species except Mycobacterium leprae. NTM widely exists in water, soil and aerosol in the natural environment, most of which are saprophytic bacteria, and only a small part are pathogenic to humans. Epidemiological analysis in recent years shows that the global incidence of NTM disease is increasing, and even some NTMs that were previously considered non-pathogenic have also been reported to be pathogenic. According to previous epidemiological surveys of tuberculosis in my country, the isolation rate of NTM increased from 4.9% in 1990 to 11.1% in 2000, and further increased to 22.9% in 2010. The reason for the increase in the incidence of NTM disease may be related to the increase in the number of people with low immunity, the increa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12Q1/686C12N15/11C12R1/32
CPCC12Q1/686C12Q1/689C12Q2563/107C12Q2545/114
Inventor 逄宇王玉峰黄海荣杜建李亮许绍发张洪静田建
Owner BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV
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