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Specific marker of mycobacterium tuberculosis and application thereof

A Mycobacterium tuberculosis, specific technology, applied in the field of biomedicine, can solve the problems of low detection rate, long culture time, high technical and professional quality requirements of personnel, etc., and achieve high specificity and sensitivity

Inactive Publication Date: 2013-04-24
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many diagnostic methods for MTB infection, and the most commonly used clinical method still relies on traditional sputum smear bacteriological examination, but the detection rate is low; MTB culture can be used as the "gold standard" for the diagnosis of tuberculosis, but the culture time is too long , generally 4-6 weeks, which is difficult to meet clinical needs; Bactec technology shortens the culture time, but the cost is high, and it is difficult to popularize in a short time; X-ray and CT examinations only provide imaging possibilities for diagnosis; polymerase chain reaction ( PCR) technology and other genetic diagnosis methods still have complex operation difficulties as clinical diagnosis, require high technical and professional quality of personnel, and still cannot be used for rapid diagnosis of bacillus-negative tuberculosis

Method used

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  • Specific marker of mycobacterium tuberculosis and application thereof
  • Specific marker of mycobacterium tuberculosis and application thereof
  • Specific marker of mycobacterium tuberculosis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, construction of recombinant plasmid pET28b-Rv3119

[0050] (1) Target gene primer design

[0051] Rv3119-F: 5'-CATGCCATGGCCAATGTGGTAG-3';

[0052] Rv3119-R: 5'-CTTCTCGAGTGGTCTATCGCCGAC-3';

[0053] The enzyme cutting sites are Nco I and Xho I, respectively.

[0054] (2) PCR amplification, cloning and sequence determination of the target gene

[0055] Mycobacterium tuberculosis H 37 Rv genomic DNA was used as a template, Rv3119-F and Rv3119-R were used as primers, and Taq enzyme was used to directly amplify the Rv3119 protein gene by PCR. PCR reaction conditions: 94°C pre-denaturation for 5 minutes; (94°C, 30s; 58°C, 30s; 72°C, 40s) 35 cycles; 72°C extension for 5min; 4°C storage. After the reaction, the target fragment was separated by 1% agarose gel electrophoresis, and then recovered with a DNA recovery kit (Invitrogen). Digested with Nco I and Xho I, and cloned into pET28b plasmid Nco I and Xho I restriction sites. Sequencing showed that the inse...

Embodiment 2

[0056] Example 2. Induced expression and purification of recombinant protein Rv3119

[0057] Immediately place the Eppdorf tube containing 100ul of BL21(DE3) physS competent cells from the -80°C freezer on ice. After 3-5 minutes, wait for the liquid in the tube to melt, put 0.5ul of the recombinant pET28b-Rv3119 plasmid with correct sequencing into competent cells, place it on ice for 45 minutes, put it in a water bath at 42°C, heat shock it for 90 seconds, and let it stand on ice for 3 minutes. Add 500ul of preheated LB medium without antibiotics, shake at 37°C, 220rmp, and incubate for 45-60min. Take a certain amount and spread it on a solid LB medium plate containing kanamycin, dry it at room temperature and place it upside down in a 37°C incubator for overnight cultivation. Pick clones, put them into LB liquid medium containing 50ug / ml kanamycin resistance, culture at 220rmp, 37°C to OD to about 0.6, add final concentration of 10mM IPTG, 37°C for 3h, collect IPTG-induced ...

Embodiment 3

[0058] Example 3. Using the commercialized 38kDa antigen (ImmunoDiagnostics, Inc., USA) as a control, evaluate the effect of recombinant Rv3119 antigen as a detection reagent on IgG in PPD-healthy control serum, PPD+ healthy control serum and tuberculosis patient serum

[0059] Anti-Rv3119 IgG responses in different serum samples were detected by indirect enzyme-linked immunosorbent assay. The specific steps are as follows: 96-well plate was coated with antigen Rv3119 (5 μg / ml) or 38kDa antigen (5ug / ml) overnight, the coating solution was discarded, washed 5 times with PBST, and dried. Add 300 μl of PBST containing 1% BSA to each well and incubate at 37°C for 2h. Spin dry, add 100 μl serum to be tested (1:50) diluted with PBST containing 1% BSA to each well, and incubate at 37°C for 0.5h. ) discard the serum, wash 5 times with PBST, and shake dry. Add 100 μl horseradish peroxidase-labeled goat anti-human IgG antibody diluted with PBST containing 1% BSA to each well, and incu...

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Abstract

The invention discloses a specific marker (Rv3119 recombinant protein) of mycobacterium tuberculosis and discloses an application of the Rv3119 recombinant protein in preparation a tuberculosis diagnostic reagent and a tuberculosis diagnostic kit. The Rv3119 specific antigen provided by the invention has high sensitivity in detecting phthisis, is a B cell target antigen with relatively strong activity, can be applied in preparation of rapid detection kit for the tuberculosis, and is safe and reliable.

Description

technical field [0001] The invention belongs to the field of biomedicine, and more specifically relates to a tuberculosis diagnostic reagent and its preparation method and application. Background technique [0002] Rapid and accurate diagnosis of Mycobacterium tuberculosis (MTB) infection is of great significance for the control of tuberculosis. There are many diagnostic methods for MTB infection, and the most commonly used clinical method still relies on traditional sputum smear bacteriological examination, but the detection rate is low; MTB culture can be used as the "gold standard" for the diagnosis of tuberculosis, but the culture time is too long , generally 4-6 weeks, which is difficult to meet clinical needs; Bactec technology shortens the culture time, but the cost is high, and it is difficult to popularize in a short time; X-ray and CT examinations only provide imaging possibilities for diagnosis; polymerase chain reaction ( PCR) technology and other genetic diagno...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12N15/70C12R1/19
Inventor 张舒林赵俊伟张苗苗付少刚郭晓奎孙战强
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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