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Mycobacterium tuberculosis detecting kit and application thereof

A Mycobacterium tuberculosis and reagent kit technology, applied in the field of immunoassay, can solve the problems of low detection rate, poor specificity, high false positive rate, etc., and achieve the effect of simple operation, low cost, and improved sensitivity and accuracy

Inactive Publication Date: 2014-12-24
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, serological detection has broad application prospects in the detection of tuberculosis due to its advantages of simple operation, low cost, high accuracy, operator friendliness, and simple equipment. However, the antigens currently used have poor specificity and false positives. The disadvantage of high rate, and some commercial kits have low detection rate and high false positive rate, so there is an urgent need to develop more effective specific antigens to improve the effect of serological testing

Method used

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  • Mycobacterium tuberculosis detecting kit and application thereof
  • Mycobacterium tuberculosis detecting kit and application thereof
  • Mycobacterium tuberculosis detecting kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] A test kit for detecting mycobacterium tuberculosis, which comprises the following components:

[0070] Coating solution: 0.05M carbonate buffer (Na 2 CO 3 : 1.59g; NaHCO 3 : 2.93g; add distilled water to 1L);

[0071] Washing solution: PBS solution containing 0.05% (v / v) Tween-20;

[0072] Blocking solution: washing solution containing 5% (w / v) skimmed milk;

[0073] Stop solution: 2M H 2 SO 4 ;

[0074] Antigen Rv1040c, whose sequence is shown in SEQ ID NO.2;

[0075] Antigen Rv2309A, its sequence is shown in SEQ ID NO.3.

Embodiment 2

[0077] PCR amplification of M. tuberculosis genes Rv1040c and Rv2309A:

[0078] The DNA of the total genome of H37Rv (accession number NC000962) was extracted with Mycobacterium tuberculosis (from Wuhan Medical Treatment Center), and the extraction method was as follows:

[0079] 1) Dispense 1ml of Mycobacterium tuberculosis bacteria solution into 1.5ml centrifuge tubes, and centrifuge at 12000rpm / min for 1min.

[0080] 2) Remove the supernatant, add 1ml of single distilled water, and boil on a metal heater at 100°C for 10min.

[0081] 3) Centrifuge at 12000rpm / min for 1min, absorb the supernatant, which contains the total H37Rv genome, and set aside.

[0082] Using the extracted total genome as a template, PCR was performed with primers designed according to Rv1040c (gene bank ID: 888533) and Rv2309A (gene bank ID: 3205076) to amplify the desired gene.

[0083] Among them, the primer design method is: use BioEdit software, according to the restriction sites inside the codin...

Embodiment 3

[0093] Transformation of expression vector pETEXba

[0094] (1) Eliminate the Xba I restriction site on the commercial pET28a (purchased from Novagen) vector by the method of end-filling modification (for details, refer to the usage method of T4DNA Polymerase of Takara Company), and obtain an intermediate vector named pET28a-△ X;

[0095] (2) Digest the intermediate vector pET28a-△X obtained in step (1) with restriction endonucleases NdeI and XhoI;

[0096] (3) Using the cdc62 gene of the extreme thermophilic archaea S. Solfataricus (GeneBank ID: 1455035) (Bender A, Pringle JR. Multicopy suppression of the cdc24budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1[ J]. Proceedings of the National Academy of Sciences, 1989, 86 (24): 9976-9980.) as the first mediator gene of the transformed vector. The gene was amplified in the genome of the extreme thermophilic archaea S. solfataricus by PCR (forward primer 14bEcoR-f: GCGCGG C...

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Abstract

The invention discloses a mycobacterium tuberculosis detecting kit and an application thereof. The mycobacterium tuberculosis detecting kit comprises a coating solution, a cleaning solution, a blocking solution, a stopping solution, an antigen Rv1040c and an antigen Rv2309A. The kit disclosed by the invention has the advantages of simplicity and convenience in operation and equipment, low cost, high precision, environment friendliness and the like; according to the mycobacterium tuberculosis detecting kit, the mixed antigens are used for coating, so that the problem of large individual difference caused when a single antigen is used for diagnosis is solved; in addition, the detecting effects of the mixed antigens are superior to the detecting effect of the single antigen on the aspects of sensitivity, precision and specificity. Compared with the similar kit sold on the market at present, the mycobacterium tuberculosis detecting kit disclosed by the invention has more remarkable advantages on the aspects of specificity, false positive rate or detection rate and has a wide application prospect in detecting mycobacterium tuberculosis.

Description

technical field [0001] The invention belongs to the technical field of immunodetection, and in particular relates to a detection kit for mycobacterium tuberculosis, and also relates to the application of the kit in detecting tuberculosis drugs. Background technique [0002] Tuberculosis is a serious infectious disease and one of the major public health problems facing the world today, and there have been pandemics worldwide. Its causative agent is Mycobacterium tuberculosis, which belongs to the genus Mycobacterium. With the emergence of more effective anti-tuberculosis drugs, the development of tuberculosis has been fully suppressed in the world. However, in recent years, due to the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the incidence of tuberculosis has risen again. The global tuberculosis situation There is a worsening trend. According to WHO statistics, about 1 / 3 of the world's population (about 2 billion) is infected with Mycobacteriu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/5695
Inventor 何正国张曙光
Owner HUAZHONG AGRI UNIV
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