DNA marker for detecting drug resistance of mycobacterium tuberculosis and application of DNA marker

A technology of Mycobacterium tuberculosis, drug resistance, applied in the direction of application, microorganism, plant genetic improvement, etc., to shorten the diagnosis time, improve the detection rate, save the treatment time and cost.

Active Publication Date: 2016-12-07
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, in actual studies, some PZA-resistant clinical Mtb strains do not have known pncA , rpsA and panD Gene mutation, what is the mechanism of PZA resista...

Method used

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  • DNA marker for detecting drug resistance of mycobacterium tuberculosis and application of DNA marker
  • DNA marker for detecting drug resistance of mycobacterium tuberculosis and application of DNA marker
  • DNA marker for detecting drug resistance of mycobacterium tuberculosis and application of DNA marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Discovery of PZA resistance gene

[0044] Existing research shows that pncA , rpsA and panD The gene is related to the drug resistance of Mycobacterium tuberculosis (Mtb) PZA. In the research of the clinical Mtb strains resistant to PZA, it was found that the two drug-resistant Mtb strains did not have mutations in the aforementioned genes, but there were Rv2783c Gene mutation. After genetic sequencing, it was found that the mutated Rv2783c 基因序列为5’-ATGTCTGCCGCTGAAATTGACGAAGGCGTGTTCGAGACGACCGCCACCATCGACAACGGGAGCTTTGGCACCCGGACCATCCGCTTCGAGACCGGCCGATTGGCCTTGCAGGCCGCCGGCGCGGTGGTCGCCTACCTCGACGACGACAACATGCTGCTGTCGGCGACCACCGCCAGCAAGAACCCCAAAGAACACTTCAACTTCTTCCCCCTCACGGTCGACGTCGAGGAGCGCATGTATGCGGCCGGCCGCATCCCCGGTTCGTTCTTCCGTCGCGAGGGCCGACCCTCCACCGACGCGATCCTGACCTGCCGGCTCATCGACCGCCCGCTGCGCCCGTCGTTTGTCGACGGGCTGCGCAACGAGATCCAAATCGTGGTGACGATTCTCAGCCTGGATCCGGGCGATCTCTACGACGTATTGGCGATCAACGCGGCGTCGGCGTCCACCCAGCTGGGCGGTCTGCCGTTCTCCGGGCCCATCGGCGGTGTGCGGGTGGCGCTCATCGACGGCA...

Embodiment 2

[0046] Example 2 Overexpression of mutant genes Rv2783c of recombinant Mtb resistant to PZA

[0047] To further confirm whether the resistance of the PZA-resistant Mtb strains found above is caused by genetic Rv2783cIn this example, the mutant Rv2783c was overexpressed in the normal Mtb strain (parental strain Mtb H37Rv) (the mutation site was described in Example 1), and the obtained Mtb strain was named Rv2783c (D67N), and the effect of this strain on PZA was tested. of drug resistance. At the same time, the normal Mtb strain (parental strain Mtb H37Rv) was overexpressed without mutation Rv2783c and empty-loaded Hsp60Rv as controls.

[0048] The specific experimental design is as follows: Bactec MGIT 960 was used to detect the resistance of Rv2783c (D67N) strain, Rv2783c non-mutant strain, empty vector Hsp60Rv strain, and parental strain Mtb H37Rv to PZA respectively, that is, to detect the MIC (minimum inhibitory effect of PZA) on the above strains. concentration), det...

Embodiment 3

[0052] Example 3 Wild-type Rv2783c interacts with POA

[0053] Pyrazinamide (PZA) is a prodrug that needs to be activated by the pyrazinamidase (PZase) of Mycobacterium tuberculosis (Mtb) into the active form pyrazinamide (POA) to inhibit Mtb. The mechanism of action of pyrazinamide (PZA) was studied in this example by isothermal titration calorimetry (ITC) method to study the interaction between wild-type Rv2783c and POA and PZA.

[0054] Experimental results and conclusions:

[0055] The above test results are figure 1 shown, figure 1 A is the experimental result of isothermal titration (ITC) between wild-type Rv2783c and POA. The upper part of the figure shows the original data, and the Y-axis represents the heat released per second when wild-type Rv2783c binds to POA; the lower part shows the are the heat released per injection of POA and the fitted curve, and the y-axis represents the heat released per mole per injection. It can be seen that higher concentrations of...

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Abstract

The invention discloses a DNA marker for detecting the drug resistance of mycobacterium tuberculosis and application of the DNA marker. It is found that mutant protein Rv2783cD67N has the DNA polymerase activity without dependence on a DNA template, is not suppressed by POA and has the phosphorolysis singe-stranded DNA (SS DNS) activity; besides, the mutant protein Rv2783cD67N can not be combined with POA; by means of the locus mutation, the drug resistance of mycobacterium tuberculosis on PZA can be remarkably caused, the locus can serve as a mark locus for molecular diagnosis of PZA drug resistance of mycobacterium tuberculosis, and the detection rate and the precision of mycobacterium tuberculosis with PZA drug resistance can be easily improved. The diagnosis time of patients can be effectively shortened, and the treatment time and cost of patients can be saved. The DNA marker for detecting the drug resistance of mycobacterium tuberculosis and application of the DNA marker provide a new thought for finding new and more effective tuberculosis drug resistance molecular markers.

Description

technical field [0001] The invention relates to a DNA marker for detecting drug resistance of Mycobacterium tuberculosis and its application. Background technique [0002] Pyrazinamide (PZA) is a first-line antituberculosis drug whose mechanism of action has not been fully elucidated. It is only known that PZA is a prodrug that needs to be activated by the pyrazinamidase (PZase) of Mycobacterium tuberculosis (Mtb) into the active form of POA to function. Therefore, the gene of PZase pncA Mutations can lead to Mtb resistance to PZA [1], so pncA It can be used as an important DNA molecular marker for rapid diagnosis of Mtb resistance to PZA, and it has been widely proved. However, there is a considerable amount of PZA-resistant Mtb that does not pncA Gene mutation, therefore, is presumed to be a mutation in the target of its active product POA, which leads to drug resistance. Recently, it has been reported that the ribosomal protein S1 rpsA gene [2] and the gene encodin...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N9/12C12N15/54C12R1/32
Inventor 张天宇摩西·M·恩扎瑞王邦兴刘燕刘洋刘志永
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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