Nucleotide mts 90 and use thereof in tuberculosis diagnosis

A nucleotide and purpose technology, applied in the field of molecular biology, can solve the problems of inability to identify human-type Mycobacterium tuberculosis, inability to effectively distinguish Mycobacterium voles, Mycobacterium africanum, etc., and achieves high accuracy, high specificity, The effect of improving detection efficiency

Inactive Publication Date: 2013-11-06
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Mtp40 is designed to identify human M. tuberculosis, but it cannot identify M. tuberculosis human type with deletion of mtp40 (some M. tubercul...

Method used

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  • Nucleotide mts 90 and use thereof in tuberculosis diagnosis
  • Nucleotide mts 90 and use thereof in tuberculosis diagnosis
  • Nucleotide mts 90 and use thereof in tuberculosis diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The acquisition of embodiment 1mts90

[0053] Through the analysis of the bioinformatics software ssGeneFinder, a fragment specifically present in the genomic DNA of Mycobacterium tubercμlosis (Mycobacterium tuberculosis) was found, which was named mts90 (Mycobacterium tubercμlosis specific sequence90), and then verified by the GenBank database (http: / / www.ncbi.nlm.nih.gov / BLAST / ) compared with the genomes of all microorganisms, it was preliminarily determined that it specifically exists in the genomic DNA of human Mycobacterium tuberculosis. In the whole genome of the strain MycobacteriμM tubercμlosis H37Rv (the standard strain of Mycobacterium tuberculosis) (Sequence ID: ref|NC_000962.3|), the position of mts90 is 2980771~2980860, including the non-coding region and the coding region. The non-coding part was not described, while the coding part was described as part of the coding sequence of a putative protein or integrase (Rv2659c). It has been reported that protei...

Embodiment 2

[0054] The specificity verification of embodiment 2mts90 in common microorganisms

[0055] 18 kinds of bacteria and fungi were selected for specificity verification. Reasons for selection: The selected bacteria and fungi are common clinical pathogens, which are most likely to interfere with tuberculosis detection; comprehensive coverage of Gram-positive and negative bacteria, including pathogenic bacteria, conditionally pathogenic Germs, probiotics (Clostridium butyricum), made the results more convincing. The specific experimental process is as follows:

[0056] Extract DNA templates of various strains by boiling method (TIANamp Bacteria DNA Kit, DP302): Take 100 μl TE buffer solution in a 1.5 μl EP tube, pick a small amount of well-growth colony in the tube, boil in 100°C water for 20 minutes, then immediately Place on ice for 10min. Centrifuge at 1,3000g for 2min, take the supernatant into a new 1.5μl EP tube, use it as a DNA template for PCR amplification, and store at -...

Embodiment 3

[0060] The specificity verification of embodiment 3mts90 in Mycobacterium

[0061] Using mycobacterial DNA as a template (the mycobacterial template used was donated by the China Institute for Food and Drug Control) to verify the specificity of mycobacteria

[0062] Boiling method to extract DNA templates of various strains: Take 100 μl TE buffer in a 1.5EP tube, pick a small amount of well-growth colonies in the tube, boil in 100°C water for 20 minutes, and then immediately place it on ice for 10 minutes. Centrifuge at 1,3000g for 2min, take the supernatant into a new 1.5μl EP tube, use it as a DNA template for PCR amplification, and store at -20°C.

[0063] The PCR amplification system, amplification primers and conditions are the same as in Example 2. The PCR products were electrophoresed on a 2.5% agarose gel and imaged with a gel imager, such as figure 2 shown. The results showed that, except for human-type Mycobacterium tuberculosis H37RV, there was no mts90 fragment...

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Abstract

The invention belongs to the technical field of molecular biological technique, particularly relates to nucleotide mts 90 and the use of a fragment thereof in tuberculosis diagnosis and mainly aims at providing a novel selection for authentication of human mycobacterium tuberculosis and diagnosis of tuberculosis. The sequence of the nucleotide mts 90 is shown as SEQIDNO.1. A reagent for detecting the human mycobacterium tuberculosis is provided. The main component of the reagent is the nucleotide mts 90. A kit for detecting the human mycobacterium tuberculosis is provided and mainly comprises the nucleotide mts 90. The use of the mts 90 in the detection of the human mycobacterium tuberculosis is further provided. The nucleotide mts 90 can be used for authentication of the human mycobacterium tuberculosis and the diagnosis of the tuberculosis.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to nucleotide mts90 and its application in tuberculosis diagnosis. Background technique [0002] Tuberculosis is a chronic infectious disease that seriously endangers human health. At present, about 2 billion people are infected worldwide. There are about 8 million to 10 million new tuberculosis patients every year, and about 2 million to 3 million deaths due to tuberculosis every year. According to the statistics of the World Health Organization, my country is one of the 22 countries with the most serious tuberculosis epidemic in the world, and it is also one of the 27 countries with the most serious MDR-TB epidemic in the world. At present, the annual incidence of tuberculosis in my country is about 1.3 million, accounting for 14.3% of the global incidence, ranking second in the world after India. According to the results of the fifth national tuberculosis e...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
Inventor 胥文春赵家宁尹一兵张雪梅何於娟王虹
Owner CHONGQING MEDICAL UNIVERSITY
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