Mycobacterium tuberculosis LAM (lipoarabinomannan) detection kit, preparation and use method thereof

A technology of Mycobacterium tuberculosis and detection kit, applied in the biological field, can solve the problems of cumbersome judgment, cumbersome, false positive sensitivity, etc., and achieve the effects of reducing reaction time, speeding up reaction speed, and increasing reaction chance.

Inactive Publication Date: 2015-12-09
广东国际旅行卫生保健中心(广东出入境检验检疫局口岸门诊部) +2
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AI Technical Summary

Problems solved by technology

[0004] At present, the detection methods for Mycobacterium tuberculosis mainly include smear microscopy. This method requires the detection personnel to have skilled detection skills, and the detection sensitivity of the method is low. Generally, the presence of 10 3 CFU/ml bacteria have a positive rate of 10-15%, and the bacteria cannot be accurately identified; the culture method is mainly carried out in infectious disease hospitals, and the detection cycle is long, and it generally takes 6 weeks to issue a report Although the automatic microbial identification instrument that appeared recently has greatly shortened the inspection cycle, it usually takes about 2 weeks, this still does not fundamentally solve the problem of efficiency, and there is a defect of high cost. The simple reagent cost is 120 yuan/test, And it requires large-scale equipment; serological testing methods mainly focus on the detection of Mycobacterium tuberculosis-related antibodies, because patients with tuberculosis infection have low immunity, often do not

Method used

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  • Mycobacterium tuberculosis LAM (lipoarabinomannan) detection kit, preparation and use method thereof
  • Mycobacterium tuberculosis LAM (lipoarabinomannan) detection kit, preparation and use method thereof
  • Mycobacterium tuberculosis LAM (lipoarabinomannan) detection kit, preparation and use method thereof

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preparation example Construction

[0067] A preparation method of the Mycobacterium tuberculosis LAM detection kit based on nano-immunomagnetic beads-near-infrared fluorescent labeling method, the steps are as follows:

[0068] (1) preparing a near-infrared fluorescent dye-labeled LAM monoclonal antibody;

[0069] Dilute Dylight800 (purchased from Thermofisher) 10 times with PBS buffer (pH7.4), take 1.4 μL and 20 μg rabbit anti-LAM monoclonal antibody (1 mg / ml) (purchased from Mossman Associates Inc) Light reaction was carried out for 2 hours; after the reaction, the labeled product was put into a dialysis bag and dialyzed against PBS buffer at 4°C for 4 hours. Add the final concentration of 1.5% BSA and 0.1% Tween20 to the labeled antibody solution, sodium azide 0.1‰, store at 4°C; dilute the labeled product 2000 times with PBS before use;

[0070] (2) Preparation of LAM polyclonal antibody

[0071] 1) Purification of LAM:

[0072] Inoculate MTBH37RV on Roche’s egg medium, culture at 37°C for 3 to 4 weeks, ...

specific example

[0115] Specific examples are: detection of LAM in pleural fluid

[0116] Take 5ml of pleural fluid for ultrasonic treatment. The working mode of ultrasonic lysis is: work for 5s, rest for 10s, Φ3mm horn, 60% energy, a total of 30 cycles.

[0117] Dilute the standard product to the concentrations specified in the instructions, take 50 μL of standard product, blank control and treated pleural effusion respectively, add 50 μL of solution A, and react at 37°C for 30 minutes;

[0118] Add 50 μL of solution B and react at 37°C for 15 minutes. Add 500 μL double-distilled water, let stand on the magnetic stand for 2 minutes, discard the supernatant, and repeat this step 3 times.

[0119] Add 100 μL PBS buffer solution (PH7.4) to each tube, and measure the fluorescence intensity with a portable flow injection fluorescence detector.

[0120] Result judgment

[0121] For qualitative detection, the cutoff value is twice the fluorescence intensity value of the negative control;

[0122...

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Abstract

The present invention relates to a nano immunomagnetic bead-near infrared fluorescence notation labeling method-based mycobacterium tuberculosis lipoarabinomannan (LMA) detection kit, a near-infrared fluorescence dye is used for labeling of a monoclonal antibody targeted to LAM, and nano magnetic bead surface is coated with a polyclonal antibody targeted to LAM, by use of the principle of double antibody sandwich method, a conjugate and free substances are magnetically separated, a a portable high sensitivity and low noise excitation fluorescence detector is used to detect the fluorescence intensity of the magnetic conjugate so as to detect the LAM content of a sample to be tested. The detection kit is applicable to the LAM detection in clinical and pathological specimens, and has the characteristics of being rapid, sensitive, anti-stray fluorescence interference, and long in emission fluorescence save time.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to a detection kit for mycobacterium tuberculosis lipoarabinomannan (LMA), a preparation method and a use method. technical background [0002] The invention establishes a nano-magnetic bead-near-infrared fluorescent label-detection kit for detecting LAM, a component related to mycobacterium tuberculosis. Including the near-infrared fluorescent dye labeling method of monoclonal antibodies, the polyclonal antibody coating method of nano magnetic beads and a series of related reagents. The invention uses a near-infrared fluorescent dye as a mark and adopts the principle of immunoassay to prepare a near-infrared fluorescent kit for detecting LAM double-antibody sandwich method. The fluorescence intensity of the magnetic bead-conjugate was measured using a portable flow injection fluorescence detector. In quantitative detection, the concentration of the target protein in the test sa...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/58
CPCG01N33/54326G01N33/54346G01N33/5695G01N33/582G01N2400/38
Inventor 陈晨明宏艳王小晋周艳容王冰李燕
Owner 广东国际旅行卫生保健中心(广东出入境检验检疫局口岸门诊部)
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