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Applications of mycobacterial ku protein

A technology for mycobacteria and Mycobacterium tuberculosis, applied in the fields of molecular biology and immunology, can solve the problems of cumbersome operation, non-isolation, cultivation and identification of NTM

Active Publication Date: 2022-02-11
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the cumbersome operation, many hospitals in my country do not carry out the isolation, cultivation and identification of NTM

Method used

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  • Applications of mycobacterial ku protein
  • Applications of mycobacterial ku protein
  • Applications of mycobacterial ku protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 Rapid identification methods such as mycobacterium PCR, RT-PCR, LAMP-PCR based on ku gene

[0037] According to the primer / probe design requirements of PCR, RT-PCR, and LAMP-PCR, primers were designed in the conserved region of the ku gene to identify mycobacteria in sputum specimens, bacterial cultures, and other samples, as well as Mycobacterium tuberculosis and non- Differential diagnosis of Mycobacterium tuberculosis. In the application example, two pairs of primers, ku-MIBC and ku-NTM, were designed. The sequences were Ku-MTBC-U: GGT GGTCGA CTA CCG CGA TCT T and Ku-MTBC-L: TCT TCG GGC TCG TCC AGC AAC C; Ku - NTM-U: ATGCGT TCB ATH TGG AAR GG and Ku-NTM-L: AGG CTC GCC AGR TCN TCR TCG GTG AT. Wherein, B, H, R and N are merged bases, B=G or T or C; H=A or T or C; R=A or G; N=A or T or C or G.

[0038] MTBC-specific primers were designed in the region where 55 ku genotypes of MTBC were consistent but different from NTM. Initially, 3 pairs of primers for ...

Embodiment 2

[0041] Embodiment 2 ELISA rapid diagnosis of tuberculosis based on Ku protein

[0042] 1. Cloning and induced expression of ku gene

[0043] Using the chromosomal DNA of the standard strain of Mycobacterium tuberculosis H37Rv as a template, the ku gene was amplified with primers (mku-PF: GCC GCG AATTCA TGC GAG CCA TTT GGA CGG GTT; mku-PR: ATA TAA AGC TTT CAC GGA GGC GTT GGGACG TTT) Cloned into the expression vector pET-32a(+). Inoculate the monoclonal colonies in 5 mL of LB liquid medium containing ampicillin, and cultivate to OD at 37°C and 180 rpm 600 When the value is 0.6, take 1 mL of bacterial liquid, add 1 μL of 1 mol / L IPTG, induce expression for 3 hours at 37°C, 180 rpm. After washing with PBS, SDS-PAGE identification was carried out, and the identified positive clones were stored at -80°C.

[0044] 2. Separation and purification of Mycobacterium tuberculosis Ku protein

[0045]The preserved strains were inoculated into 5 mL of LB liquid medium containing ampicilli...

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Abstract

The invention provides the application of mycobacterial Ku protein. The invention provides a new marker, the ku gene / protein belonging to the NHEJ system, which can be used for the identification of mycobacteria and the differential identification of MTBC and NTM. The ubiquity of ku gene in mycobacteria and the rarity in prokaryotic cells determine the applicability and high specificity of the detection technology based on this marker to the entire genus of mycobacteria. Based on the ku gene / protein, rapid identification methods such as PCR, RT-PCR, LAMP-PCR, enzyme-linked immunoassay (ELISA), and immune colloidal gold can be established and corresponding reagents can be developed. The detection reagent based on the invention can be widely used in related fields such as auxiliary diagnosis of tuberculosis, epidemiological monitoring and the like.

Description

technical field [0001] The invention relates to the fields of molecular biology and immunology, in particular to the application of mycobacterial Ku protein. Background technique [0002] Mycobacteria include Mycobacterium tuberculosis complex (MTBC) and Non-tuberculosis mycobacteria (NTM), so far there are nearly 160 species and subspecies. Mycobacteria are acid-fast stain-positive, gram-positive bacilli, many species of which can cause serious disease in humans and animals. Among them, tuberculosis is still one of the ten deadliest diseases in the world. At the same time, the number of "tuberculosis" caused by non-tuberculous mycobacterium infection is also rising rapidly. 25-50% of AIDS patients are combined with non-tuberculous mycobacterium infection, which seriously increases the difficulty of treatment. [0003] The culture conditions and clinical treatment drugs of mycobacteria are different from other bacteria. All traditional experiments based on the growth of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/68A61K39/04A61K39/40A61P31/06C12Q1/689C12Q1/04C12N15/11C12R1/32
CPCG01N33/5695G01N33/6893A61K39/04A61K39/40A61P31/06C12Q1/689A61K2039/53
Inventor 王瑞白万康林刘海灿
Owner ICDC CHINA CDC
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