Method and kit for detecting streptomycin medicine resistant mutation of Mycobacterium tuberculosis

A technology for Mycobacterium tuberculosis and Bacillus streptomycin, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, material excitation analysis, etc. question

Inactive Publication Date: 2011-11-02
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the detection rate and throughput of these methods are different, the common disadvantage is that PCR post-processing is required, which not only affects t...

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  • Method and kit for detecting streptomycin medicine resistant mutation of Mycobacterium tuberculosis
  • Method and kit for detecting streptomycin medicine resistant mutation of Mycobacterium tuberculosis
  • Method and kit for detecting streptomycin medicine resistant mutation of Mycobacterium tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1 Primer and probe design

[0080] Multiple pairs of primers and probes were designed at each of the four sites according to the wild-type genome sequence of Mycobacterium tuberculosis, and there was no complementary sequence at the 3' end of the primers. The combination of primer and probe sequence can be:

[0081] Primer rpsL43F:

[0082] 5′-CAGCCCGCAGCGTCGTGGTGT-3′

[0083] Primer rpsL43R:

[0084] 5′-GTGGCCCTCGCCGGGAA-3′

[0085] Primer rpsL88F:

[0086] 5′-GCACTCGATGGTGCTGGTGC-3′

[0087] Primer rpsL88R:

[0088] 5′-CGTGCCTGTTTGCGGTTCTT-3′

[0089] Primer rrs512F:

[0090] 5′-ACCTCTTTTCACCATCGACGAAGGTCC-3′

[0091] Primer rrs512R:

[0092] 5′-GTTAAGCCGTGAGATTTCACGAACAA-3′

[0093] Primer rrs904F:

[0094] 5′-GGGTTTCTCTTCCTTGGGATCCGTG-3′

[0095] Primer rrs904R:

[0096] 5′-GCATGTCAAACCCAGGTAAGGTTC-3′

[0097] Probe rpsL43P:

[0098] 5′-FAM-CCGCGCCACTCCGAAGAAGCCGAACTCCGCGG-Dabcyl-3′

[0099] Probe rpsL88P:

[0100] 5′-TET-CCGTCCGGGTGAAGG...

Embodiment 2

[0106] Example 2 Extraction of Mycobacterium tuberculosis Genomic DNA

[0107] The genomic DNA of Mycobacterium tuberculosis is extracted by thermal cracking method, and the specific process is as follows:

[0108] A. For Mycobacterium tuberculosis growing on solid medium, use 22SWG standard inoculation loop to collect 1 loop of bacteria, and suspend in 250 μL TB DNA extraction solution. Take 1 mL of Mycobacterium tuberculosis grown in liquid medium, centrifuge at 10,000 rpm for 15 min, discard the supernatant and resuspend the bacteria in 250 μL of TB DNA extract.

[0109] B. Seal with parafilm and heat at 99°C for 20 minutes. Centrifuge at 14000rpm for 10min, transfer the supernatant to a new 1.5mL centrifuge tube. The supernatant is the template for PCR amplification.

[0110] C. Samples should be stored at -20°C and the test should be completed within 1 month. Be careful not to repeatedly freeze and thaw samples.

Embodiment 3

[0111] The establishment and optimization of embodiment 3 reaction system

[0112] The wild-type Mycobacterium tuberculosis H37Rv genomic DNA was used as the sample to be tested, and stored at -20°C after aliquoting.

[0113] 2.1 Optimization of the primer ratio: under the condition that other conditions in the reaction system are the same, adjust the ratio of the upstream and downstream primers to 1:5, 1:10, 1:15 and 1:20 respectively. Set 1:10 as the ratio of upstream and downstream primers.

[0114] 2.2MgCl 2 Concentration optimization: under the same conditions in the reaction system, the MgCl 2 The concentration of the magnesium ion increases from 1mmol / L to 5mmol / L in 1mmol / L increments. After repeated experiments, 1mmol / L (rpsL system) and 3mmol / L (rrs system) are selected as the magnesium ion concentration in the reaction system of the kit.

[0115] 2.3 Optimization of the amount of Taq enzyme: By comparing the optimization experiment results of the amount of Taq en...

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Abstract

The invention discloses a method and a kit for detecting the streptomycin medicine resistant mutation of Mycobacterium tuberculosis. The invention relates to medicine resistant mutation detection technique and provides a method for detecting streptomycin medicine resistant mutation of Mycobacterium tuberculosis, which effectively improves sensitivity and specificity, and is simple and convenient in operation and short in period. The method comprises: designing primers and probes according to the complete sequence of the Mycobacterium tuberculosis and the gene sequences of the genomes rpsL and rrs of the Mycobacterium tuberculosis; extracting the DNA of a sample of the Mycobacterium tuberculosis; constructing a polymerase chain reaction (PCR) reaction system; and performing PCR amplification and analysis on a fusion curve. In the method, experiments are performed in two tubes respectively by using the specific primers and probes, and the amplification of the nucleic acid fragment of a target nucleotide sequence and subsequent analysis on the fusion curve are realized by using heat-resistance DNA polymerase, four kinds of nucleotide monomers and other components and by using real-time PCR technique. A fluorescent PCR fusion curve method with high specificity can quickly and accurately detects common medicine-resistance mutation of Mycobacterium tuberculosis and is expected to be directly used for medicine-resistance detection of a clinic Mycobacterium tuberculosis sample.

Description

technical field [0001] The invention relates to the detection technology of drug-resistant mutations, in particular to a probe melting curve analysis technology based on double-labeled self-quenching probes for detecting streptomycin drug-resistant mutations of Mycobacterium tuberculosis. Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis infection. Mycobacterium tuberculosis may invade various organs of the human body, but mainly invades the lungs, which is called pulmonary tuberculosis. By the late 1980s, due to factors such as poverty, AIDS, population mobility, and drug resistance, the incidence and mortality of tuberculosis rose rapidly around the world, becoming the most serious infectious disease in the world alongside AIDS. The drug resistance of Mycobacterium tuberculosis is serious, and the drug resistance rate is as high as 46%, which poses a severe challenge to the prevention and treatment of tuberculo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02G01N21/64
Inventor 李庆阁张婷胡思玉
Owner XIAMEN UNIV
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