Mycobacterium tuberculosis mutant gene and application thereof

A technology of Mycobacterium tuberculosis and mutant gene, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of ineffective treatment, need to improve sensitivity and specificity, increase adverse drug reactions, etc., and achieve strong sensitivity and safety. specific effect

Pending Publication Date: 2020-08-25
AFFILIATED HOSPITAL OF ZUNYI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, Mycobacterium tuberculosis (Mtb) has cross-resistance to aminoglycoside anti-tuberculosis drugs, and improper use of antibiotics may lead to ineffective treatment and increase adverse drug reactions
At present, there are molecular susceptibility detection techniques for aminoglycoside anti-tuberculosis drugs. For the detection of rrs or rpsl gene mutations, the detection rate of STR drug-resistant bacteria can reach 75% to 90%, but the detection rate of AMI and KAN drug-resistant bacteria is only 5% to 78%, sensitivity and specificity need to be improved
Moreover, the existing molecular drug sensitivity detection technology of aminoglycoside anti-tuberculosis drugs cannot directly guide clinical medication, so it is difficult to improve the cure rate

Method used

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  • Mycobacterium tuberculosis mutant gene and application thereof
  • Mycobacterium tuberculosis mutant gene and application thereof
  • Mycobacterium tuberculosis mutant gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] The Rv1527c gene fragment of the laboratory standard strain H37Rv was knocked out by using the phage-mediated gene knockout technology of Mycobacterium tuberculosis, and the Rv1527c gene knockout strain H37RvΔRv1527c of Mycobacterium tuberculosis was successfully constructed, and the function of the Rv1527c gene was further studied. Design primers for the left (L) and right (R) arms of the Rv1527c gene, namely Rv1527c-LFP (5'-TTTTTTTTCCATAAATTGGTTCAGCGACGACGGCCT-3'), Rv1527c-LRP (5'-TTTTTTTTCCATTTCTTGGACCCCATACCAATGACAGCTA-3') and Rv1527c-RFP (5'- TTTTTTTCCATAGATTGGATCCGCATTTCCGCCAC-3'), Rv1527c-RRP (5'-TTTTTTTTCCATCTTTTGGGGCGGTAGGCCCAGTTCA-3'), PCR amplification of Rv1527c gene L, R arm, construction of homologous exchange site (AES); then integrated into the Mycobacterium tuberculosis phage genome , to obtain phagemids; introduce phagemids into Mycobacterium smegmatis, amplify in vitro to obtain high-titer recombinant phages, transfect Mycobacterium tuberculosis and sm...

Embodiment 2

[0019] The minimum inhibitory concentration (MIC) test was conducted on the H37Rv standard bacteria and the H37Rv△Rv1527c knockout bacteria of Mycobacterium tuberculosis against 12 commonly used anti-tuberculosis drugs.

[0020] Use TREK produced by Thermo Fisher MYCOTB susceptibility plate ("MYCOTB" for short), containing the minimum inhibitory concentration (minimum inhibitory concentration, MIC) susceptibility plate of 12 kinds of anti-tuberculosis drug freeze-dried powder, including ofloxacin, moxifloxacin, rifampicin , amikacin, streptomycin, rifabutin, p-aminosalicylic acid, ethionamide, cycloserine, isoniazid, kanamycin and ethambutol. The drug susceptibility plate uses American Type Culture Collection (ATCC) H37Rv (ATCC 27294) and H37Ra (ATCC 25177) for quality control to ensure the effective activity of the drugs on the drug susceptibility plate.

[0021] The critical concentration for drug susceptibility testing of Mycobacterium tuberculosis refers to the American ...

Embodiment 3

[0029] For the mutant bacteria containing the Rv1527c A1380P mutant gene and the Rv1527c G1637A mutant gene in the clinical isolate of Mycobacterium tuberculosis, the minimum inhibitory concentration (MIC) test of the amikacin antibiotic was carried out according to the method of Example 1. Both mutants showed strong sensitivity to amikacin antibiotic.

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Abstract

The present invention belongs-the technical field of biomedicines and provides a mycobacterium tuberculosis mutant gene and an application thereof. The mutant gene is a mutant gene in which an Rv1527cgene fragment is deleted in a H37Rv gene of a standard strain of mycobacterium tuberculosis, or a mutant gene with an alanine at 1380th position on the Rv1527c gene fragment of the mycobacterium tuberculosis is mutated to proline or glycine at 1637th position is mutated to alanine. The mutant gene is used as a biomarker of the mycobacterium tuberculosis for detection of clinical specimens of tuberculosis patients before treatment and conducive-clinical formulation of reasonable chemotherapy regimens, and thus improves a clinical successful treatment rate of tuberculosis patients, especially multidrug-resistant tuberculosis patients.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a mutant gene of mycobacterium tuberculosis and its application. Background technique [0002] Aminoglycosides are injectable anti-tuberculosis drugs recommended by the World Health Organization, including streptomycin (STR), amikacin (AMI) and kanamycin (KAN). STR is mainly used for the treatment of common tuberculosis, while AMI and KAN are currently used for the treatment of multidrug-resistant tuberculosis (MDR-TB, which means that Mycobacterium tuberculosis is resistant to both rifampin and isoniazid anti-tuberculosis drugs). Core second-line drugs. STR, AMI and KAN act on the ribosome of Mycobacterium tuberculosis (Mtb), interfere with the normal synthesis of bacterial proteins, and have strong bactericidal activity in vitro. It is known that rrs, rpsL, eis promoter and tlyA gene mutations are related to the resistance of Mycobacterium tuberculosis to aminoglycosides....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12Q1/689C12R1/32
CPCC12N9/1029C12Q1/689C12Q2600/156C12Q2600/106
Inventor 陈玲赵昭亮刘梅李青彭章丽李娜娜李秋阳徐鹏张建勇
Owner AFFILIATED HOSPITAL OF ZUNYI UNIV
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