A dna marker for detecting drug resistance of mycobacterium tuberculosis and its application

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: 2019-08-27
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • 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 resistance in these clinical Mtb strains? How can it be detected more conveniently and improve the detection rate of PZA resistance of Mycobacterium tuberculosis? is still a problem

Method used

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  • A dna marker for detecting drug resistance of mycobacterium tuberculosis and its application
  • A dna marker for detecting drug resistance of mycobacterium tuberculosis and its application
  • A dna marker for detecting drug resistance of mycobacterium tuberculosis and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Discovery of PZA drug-resistant genes

[0044] Existing research shows that, pncA , rpsA and panD The gene is related to the PZA drug resistance of Mycobacterium tuberculosis (Mtb). In the study of the clinical Mtb strains resistant to PZA, the present invention found that the above-mentioned gene mutations did not occur in the two drug-resistant Mtb strains, but there were Rv2783c Genetic mutations. After gene sequencing, it was found that the mutated Rv2783c 基因序列为5’-ATGTCTGCCGCTGAAATTGACGAAGGCGTGTTCGAGACGACCGCCACCATCGACAACGGGAGCTTTGGCACCCGGACCATCCGCTTCGAGACCGGCCGATTGGCCTTGCAGGCCGCCGGCGCGGTGGTCGCCTACCTCGACGACGACAACATGCTGCTGTCGGCGACCACCGCCAGCAAGAACCCCAAAGAACACTTCAACTTCTTCCCCCTCACGGTCGACGTCGAGGAGCGCATGTATGCGGCCGGCCGCATCCCCGGTTCGTTCTTCCGTCGCGAGGGCCGACCCTCCACCGACGCGATCCTGACCTGCCGGCTCATCGACCGCCCGCTGCGCCCGTCGTTTGTCGACGGGCTGCGCAACGAGATCCAAATCGTGGTGACGATTCTCAGCCTGGATCCGGGCGATCTCTACGACGTATTGGCGATCAACGCGGCGTCGGCGTCCACCCAGCTGGGCGGTCTGCCGTTCTCCGGGCCCATCGGCGGTGTGCG...

Embodiment 2

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

[0047] In order to further confirm whether the drug resistance of the PZA-resistant Mtb strains found above is caused by genes Rv2783c Due to the mutation, this example overexpresses the mutated Rv2783c in the normal Mtb strain (parent strain Mtb H37Rv) (the mutation site is as described in Example 1), and the resulting Mtb strain is named Rv2783c (D67N), and the strain is tested for PZA drug resistance. At the same time, normal Mtb strains (parental strain Mtb H37Rv) were overexpressed in vivo without mutation Rv2783c, and empty Hsp60Rv were used as controls.

[0048]The specific experimental design is as follows: use Bactec MGIT 960 to detect the drug 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 bacteriostatic factor) of PZA to the above ...

Embodiment 3

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

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

[0054] Experimental results and conclusions:

[0055] The above test results are as figure 1 as shown, figure 1 A is the result of the isothermal titration (Isothermal titration calorimetry, ITC) experiment between wild-type Rv2783c and POA. The upper part of the figure shows the original data, and the Y-axis indicates the heat released per second when wild-type Rv2783c binds to POA; the lower part shows is the heat released per injection of POA and the fitted curve, and the Y axis represents the heat released per mole per inj...

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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 anti-tuberculosis drug, and its mechanism of action has not been fully elucidated. At present, it is only known that PZA is a prodrug that needs to be activated by pyrazinamidase (PZase) of Mycobacterium tuberculosis (Mtb) into the active form POA to work. Therefore, the PZase gene pncA Mutations can cause Mtb to be resistant to PZA[1], so pncA It can be used as an important DNA molecular marker to quickly diagnose whether Mtb is resistant to PZA, and has been widely proven. However, a considerable number of PZA-resistant Mtb did not pncA Genetic mutations, therefore, presumably mutate the target of its active product, POA, leading to drug resistance. Recently, it has been reported that the protein encoding ribosomal protein S1 rpsA Gene[2] and the gene encoding as...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N9/12C12N15/54C12R1/32
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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