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ELISpot tuberculosis infection diagnostic reagent kit and its application

A diagnostic kit and a technology for tuberculosis, applied in the field of medical immunology diagnosis, can solve the problems of high detection cost, high cost of the kit, difficulty in widespread application and the like, and achieve the effects of simple preparation and low cost

Inactive Publication Date: 2008-07-16
中国人民解放军总医院第二附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This experiment is not affected by BCG vaccination and environmental mycobacterial infection, and its detection sensitivity and specificity are high, but it uses a series of synthetic peptides of two antigens as stimulating antigens, which makes the cost of the kit high and the detection cost high. It is difficult to widely apply

Method used

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  • ELISpot tuberculosis infection diagnostic reagent kit and its application
  • ELISpot tuberculosis infection diagnostic reagent kit and its application
  • ELISpot tuberculosis infection diagnostic reagent kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] One, a kind of preparation method of ELISpot tuberculosis infection diagnostic kit:

[0075] (1) Preparation of CFP10-ESAT6 fusion protein by genetic engineering technology:

[0076] 1. Primer design and synthesis

[0077] cfp10 primer design:

[0078] P1 (upstream): 5'-CCGGATCCATGGCAGAGATGAAGAC-3'

[0079] P2 (downstream): 5'-GCTGCCGCCACCGCCGCTTCCGCCACCGCCGCTTCCACCGCCACCGAAGCCCATTTGCGAGGACAGCGCCT-3'

[0080] Amplified fragment: 353bp

[0081] esat6 primer design:

[0082] P3 (upstream): 5'-GGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGCAGCATGACAGAGCAGCAGTGGAATTTCGCGG-3'

[0083] P4 (downstream): 5'-CC AAGCTT TGCGAACATCCCAGTGA-3'

[0084] Amplified fragment: 338bp

[0085] 2. PCR amplification of cfp10 and esat6 genes

[0086] Using primers P1, P2, P3, and P4 respectively, under the action of Taq plμs I DNA polymerase, the CFP10 and ESAT6 genes were amplified using the Mycobacterium tuberculosis H37Rv genomic DNA as a template. PCR reaction program: 95°C for 5min; 9...

Embodiment 2

[0176] Application of ELISpot Tuberculosis Infection Diagnostic Kit for detection:

[0177] The ELISpot tuberculosis infection diagnostic kit provided by the present invention was used to detect Wang, female, 36 years old, hospitalized patient from the Gastroenterology Department of the Second Affiliated Hospital of the PLA General Hospital, hospital number: 241298, diagnosis: incomplete pyloric obstruction.

[0178] (1) Coating:

[0179] 1. Add 100 μL / well of IFN-r capture antibody diluted 1:60 to a 96-well filter plate, cover it, and incubate at 37°C for 1 hour;

[0180] 2. Discard the coating solution and wash the plate 3 times with sterile phosphate buffered saline (PBS), 3 min each time;

[0181] 3. Add 200 μL of PBS containing 3% bovine serum albumin to each well, cover and incubate at 37°C for 2 hours;

[0182] 4. Discard the supernatant, put the plate in a sealed bag, and store it at 4°C for 1 day;

[0183] (2) Detection:

[0184] 1. Take 5ml of blood and add it to...

Embodiment 3

[0222] Application of ELISpot Tuberculosis Infection Diagnostic Kit for detection:

[0223] The ELISpot tuberculosis infection diagnostic kit was used to detect Wu, an 81-year-old inpatient from the Neurology Department of the Second Affiliated Hospital of the PLA General Hospital, hospital number: 108764. Diagnosis: 1. Cerebral infarction; 2. Hypertension grade III.

[0224] (1) Coating:

[0225] 1. Add 100 μL / well of IFN-r capture antibody diluted 1:60 to a 96-well filter plate, cover it, and incubate at 37°C for 2 hours;

[0226] 2. Discard the coating solution and wash the plate 3 times with sterile phosphate buffered saline (PBS), 3 min each time;

[0227] 3. Add 200 μL of PBS containing 2% bovine serum albumin to each well, cover and incubate at 37°C for 2 hours;

[0228] 4. Discard the supernatant, put the plate in a sealed bag, and store at 4°C for 1 week;

[0229] (2) Detection:

[0230] 1. Take 4ml of blood and add it to the blood collection tube containing hepar...

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Abstract

The invention relates to an ELISpot tuberculosis infection diagnostic kit and the application, which adopts the genetic engineering technology to obtain a CFP10-ESAT6 fusion protein antigen from a mycobacterium tuberculosis by cloning, expression and purification, a tuberculosis specific cell stimulator is used for stimulating the peripheral blood T lymphocyte cell of the detected person to secrete a specific IFN-Gamma, then the invention is detected by ELISpot, and the whole process needs to take two days. The usage of the invention can be used for detecting whether the detected person is infected by mycobacterium tuberculosis by using the naked eye or instrument to determine the result according to the number of the generated purple blue spots, so as to assist the diagnosis of tuberculosis and differential diagnosis.

Description

technical field [0001] The invention relates to an ELISpot tuberculosis infection diagnostic kit and an application thereof, belonging to the technical field of medical immunology diagnosis, in particular to an ELISpot tuberculosis infection diagnostic kit and its application in the detection of tuberculosis infection. Background technique [0002] Tuberculosis is still one of the major infectious diseases that endanger human health in the world. Since 1985, the prevalence of AIDS, tuberculosis-infected immigrants and some people living in poverty have caused the incidence of tuberculosis to rise in the United States and other developed countries in Europe and the United States, especially tuberculosis. The problem of bacterial drug resistance and the incidence of non-tuberculous mycobacterial disease are increasing year by year, which makes the treatment of tuberculosis even worse. At present, there are about 20 million tuberculosis patients in the world, 8 to 10 million ne...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N1/28
Inventor 吴雪琼李峤坷阳幼荣梁艳张俊仙李娟
Owner 中国人民解放军总医院第二附属医院
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