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Kit for detecting sensitive mycobacterium tuberculosis and detection method of kit

A technology for detecting Mycobacterium tuberculosis and a detection method, which is applied in the field of kits for detecting sensitive Mycobacterium tuberculosis, can solve problems such as difficulty in extracting Mycobacterium DNA, inability to distinguish antibodies, etc., and achieve good practicability

Inactive Publication Date: 2019-04-19
ANHUI UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method cannot distinguish between natural infection and antibodies caused by vaccination. Spanish scholars used the PCR method to detect tuberculosis from confirmed cases (Parra et al., 2008), and the detection rate was only 80.64%
However, this method is very harsh on the pre-treatment of the sample, and it is very difficult to extract mycobacterial DNA from the sample; in addition, the GC content of the mycobacterial genome is as high as 66%, and it is also difficult to amplify the target fragment itself

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Preparation of Enzyme-Linked Immunosorbent (ELISA) Reagent for Detecting Tuberculosis Antibody

[0058] 1). Dilute the purified recombinant protein of Rv0899 or Rv1973 or Mce4B with carbonate buffer to a concentration of 3ng-6μg / μL, and coat a 96-well microtiter plate with 90μL per well; react at 37°C for 50 minutes; The carbonate buffer solution is 8mmNa2CO3 and NaHCO3 solution, pH9;

[0059] 2). Use 3.5-4.5% casein, bovine serum albumin, gelatin or skimmed milk powder to seal the microplate, 200 μL per well; react at 37°C for 50 minutes;

[0060] 3). Washing 3 times with PBST, said PBST is a mixture of 60mM Na2HPO4, 15mM NaH2PO4, 80mM NaCl, 0.12% Tween-20, its pH7.3-7.4;

[0061] 4). Vacuum-sterile and dry-pack the ELISA plate. Store in low-temperature refrigeration, and the storage period is more than half a year; operate according to the instructions when using, as described in steps 5-9; the kit can detect tuberculosis antibodies in various serum samples;

[006...

Embodiment 2

[0068] Preparation of Colloidal Gold Test Strip Reagent for Detecting Tuberculosis Antibody

[0069] 1). Add 1-1.5% sodium citrate to 0.02% chloroauric acid (HAuCl4), stir while adding, and cook at 90°C for 20 minutes to obtain a colloidal gold solution with 20-30nm particles;

[0070] 2). Preparation of recombinant staphylococcal protein A / G and colloidal gold markers: Use 0.02-0.0.09M potassium carbonate to adjust the colloidal gold solution cooled to room temperature to pH 5.5-6, slow down the recombinant staphylococcal protein A / G Slowly add to the colloidal gold solution and mix well, and react at room temperature for 0.6-0.8h. Then, bovine serum albumin with a final concentration of 0.08% was added to the reaction solution as a stabilizer, and concentrated by high-speed centrifugation at 4° C. for 25 minutes. Wash 4-5 times with 0.08% BSA to obtain purified recombinant staphylococcus protein A / G and colloidal gold marker. Suspend the marker in 3-6mm of 0.08% BSA and 0....

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Abstract

The invention relates to the technical field of detection, in particular to a kit for detecting sensitive mycobacterium tuberculosis and a detection method of the kit. The kit is used for testing mycobacterium tuberculosis, and the preparation steps of a reagent include putting a sputum sample into a sodium hydroxide solution, and shaking and removing mucin components on the surface of the sputumsample; preforming high-speed centrifugation on the sputum sample to extract required mycobacterium tuberculosis cells; crushing the extracted Mycobacterium tuberculosis cells, centrifuging, washing with distilled water, and dissolving the obtained precipitate with a membrane dissolving solution to obtain an outer membrane protein; separating membrane outer proteins to obtain three outer membraneproteins, and purifying and recombining the three outer membrane proteins to obtain three recombined proteins; diluting the purified three recombined protein solution and using an enzyme label plate;closing the enzyme label plate and washing; and putting the enzyme label plate into a vacuum aseptic dryer for drying and then putting into a reagent bottle. The method can detect an antibody in the sample and a source of the antibody.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a kit for detecting sensitive Mycobacterium tuberculosis and a detection method thereof. Background technique [0002] China is one of the 22 countries with a high burden of tuberculosis in the world, and the annual new cases account for 16% of the global total. China's tuberculosis control work directly affects the implementation and results of the global tuberculosis control strategy. According to WHO's estimate, there are 1.3 million cases of active pulmonary tuberculosis patients in my country every year, and my country has become one of the countries with high burden of tuberculosis in the world, ranking second among the 22 countries with high burden of tuberculosis. Nearly half (550 million) of the country's population is infected with tuberculosis, which is significantly higher than the level of 1 / 3 of the global population; there are 5 million cases of active pulmonary...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/558
CPCG01N33/543G01N33/558G01N33/5695G01N2469/20
Inventor 王晓春张延鹏李辉
Owner ANHUI UNIV OF SCI & TECH
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