Diagnostic reagent of tuberculosis and kit

A diagnostic reagent and technology for tuberculosis, applied in the field of diagnostic reagents and kits containing Mycobacterium tuberculosis Rv1985c protein, can solve the problems of weakened diagnostic value and achieve the effects of improving diagnostic sensitivity, simple assay, and low cost

Active Publication Date: 2010-03-03
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the presence of pure protein derivatives of M. tuberculosis cross-reacts with environmental mycobacteria and the tuberculosis vaccine (M.bovis BCG) strain, which weakens its diagnostic value

Method used

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  • Diagnostic reagent of tuberculosis and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037]Embodiment 1 prepares tuberculosis detection reagent and test kit

[0038] The kit of the present invention contains the recombinant antigen Rv1985c and its specific antibody, and also contains various reagents and utensils for detection, including various buffers, diluents, reaction solutions and stop solutions, specifically including the following reagents and articles :

[0039] 1) Coating buffer (0.05M carbonate buffer), pH 9.6: 1.59g Na 2 CO 3 , 2.93 g NaHCO 3 .

[0040] 2) Wash solution (PBST), pH 7.4: 8g NaCl, 0.2g KCl, 0.2g KH 2 PO 4 , 2.9 g Na 2 HPO 4 12H 2 O, 0.5ml Tween-20, 1L H 2 O.

[0041] 3) Blocking solution (0.1% BSA / PBS), pH7.4: 8g NaCl, 0.2g KCl, 0.2g KH 2 PO 4 , 2.9gNa 2 HPO 4 12H 2 O, 0.1g BSA (Bovine Serum Albumin), 1L H 2 O.

[0042] 4) Substrate buffer, 0.2M NaH 2 PO 4 (28.4 g / L) 25.7ml, 0.1M citric acid (19.2 g / L) 24.3ml, add distilled water 50ml.

[0043] 5) Chromogenic solution: 20ml substrate buffer, 8mg OPD, 25ul 30%H 2 o...

Embodiment 2E

[0047] Embodiment 2ELISA test detects pulmonary tuberculosis

[0048] The sample to be tested is serum. Whole blood is collected and added with 2.0 mg / ml EDTA anticoagulant, centrifuged at 3000 rpm for 10 min, and the supernatant is collected. When saving, it can be divided into cryopreservation tubes and stored in a -70 degree refrigerator. The samples in this example were divided into two groups: tuberculosis patients and healthy controls.

[0049] Tuberculosis patients were recruited from Shandong Provincial Pulmonary Hospital in Jinan and Pulmonary Hospital in Chongqing from May 2007. Part of the healthy controls came from the above-mentioned hospitals (non-tuberculosis patients or medical staff), and the other part were non-tuberculosis patients or medical staff recruited at Fudan University and Shanghai Huashan Hospital in April 2008.

[0050] The inclusion criteria for the two groups of people are as follows:

[0051] Pulmonary tuberculosis patients: clinical sputum ...

Embodiment 3

[0078] Example 3 Colloidal gold label detection

[0079] 1. Take 100ml of 0.01% HAuCl4 aqueous solution, add 2ml of 1% trisodium citrate aqueous solution, heat and boil for 15min to 30min, until the color turns red and does not change.

[0080] 2. Mix the Rv1985c antigen with the prepared colloidal gold at a ratio of 1:2, add BSA with a final concentration of 1% as a stabilizer, and add sodium azide with a final concentration of 0.1% as a preservative.

[0081] 3. Add 10ul of Rv1985c antigen to the nitrocellulose membrane and let it stand overnight.

[0082] 4. Add blocking solution (1% BSA solution) for 5 minutes the next day

[0083] 5. Add the diluted serum to be tested, which can be diluted 1:10

[0084] 6. After washing with PBS, add the above-mentioned labeled colloidal gold antigen

[0085] 7. Interpret the result after reacting for 5-15 minutes. By visual observation, if there is a red band, it is judged as positive, otherwise it is negative.

[0086] The results s...

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Abstract

The invention belongs to the field of diagnostic reagents, and relates to a diagnostic reagent containing mycobacterium tuberculosis Rv1985c protein and a kit. The diagnostic reagent and the kit can perform rapid diagnosis on whether the blood or the body fluid of an experimenter is infected by the mycobacterium tuberculosis in the time periods from 15 minutes of an immune colloidal gold to 5 hours of ELISA. When the diagnostic reagent is used for the clinically-proved abortive tuberculosis, a Rv1985c antigen is used singly to detect that the sensitivity reaches 59 percent and the specificityreaches 96 percent; and when used together with other diagnostic antigens (such as a control antigen LAM / 38kDa), the diagnostic reagent can further improve the diagnostic sensitivity to 75 percent and has high clinical application values. The detection with the diagnostic reagent only needs a single blood serum sample, is simple and quick in test, needs no specialized laboratory equipment, is lowin cost, offers test results in the same day, and is very suitable for the detection of tuberculosis infection in extensive rural hospitals of villages and towns and under the condition of battlegrounds.

Description

technical field [0001] The invention belongs to the field of diagnostic reagents, and relates to a tuberculosis diagnostic reagent and a kit, in particular to a diagnostic reagent and a kit containing Mycobacterium tuberculosis Rv1985c protein. Background technique [0002] Tuberculosis is one of the major public health problems facing the world today. Its pathogen is Mycobacterium tuberculosis, which belongs to the genus Mycobacterium. According to reports, there are about 8-10 million new cases of tuberculosis in the world every year, and 2-3 million people lose their lives every year. Tuberculosis has become the number one infectious disease causing death among adults. According to WHO reports, one-third of the world's population (about 2 billion) has been infected with Mycobacterium tuberculosis. If no measures are taken, about 300 million people will be infected by Mycobacterium tuberculosis in the past 10 years. According to the fourth national epidemiological survey...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/53G01N33/543
Inventor 张文宏陈嘉臻王森苏晓迪张颖王洪海张舒邵凌云
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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