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Specific electrochemical immunosensor for tuberculosis serodiagnosis

An immunosensor and electrochemical technology, applied in the direction of material electrochemical variables, chemical instruments and methods, scientific instruments, etc., can solve the problem that diagnostic kits cannot be recommended for tuberculosis diagnosis

Inactive Publication Date: 2011-09-21
SOUTHWEST UNIVERSITY
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Problems solved by technology

However, in 2008, WHO / TDR investigated 19 rapid tuberculosis detection kits that are currently commercially available in the same laboratory, and their sensitivity and specificity were found to be between 0.97% and 0.97% respectively based on the results of mycobacterial culture and clinical follow-up. -59.7% and 53%-98.7%, emphasizing that these diagnostic kits cannot be recommended for TB diagnosis

Method used

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  • Specific electrochemical immunosensor for tuberculosis serodiagnosis
  • Specific electrochemical immunosensor for tuberculosis serodiagnosis

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Embodiment Construction

[0011] 1. Cloning of Rv2175c gene

[0012] Using primer pair 2715a (5'-AGCCTA GGATCC ATGCCTGGCC-3', the underlined part is BamHI restriction site) and 2715b (5'-GAG AAGCTT CTGCAACCCCGGT-3'; the underlined part is the HindIII restriction enzyme site) the open reading frame Rv2175c was amplified from the Mycobacterium tuberculosis H37Rv genome;

[0013] The amplified product was digested with BamHI and HindIII, and connected to the pET28a plasmid vector digested with the same restriction enzymes;

[0014] The recombinant plasmid pET28a-rv2175c was obtained after transformation of Escherichia coli DH5α was identified by PCR and verified by sequencing (sequencing results showed that the aspartic acid at position 21 of the protein was mutated into glutamic acid, which is not mentioned in the current research literature. This protein plays a special function, so continue to the next step of experimental research).

[0015] 2. Expression and purification of protein encoded by Rv...

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Abstract

The invention relates to a specific electrochemical immunosensor for tuberculosis serodiagnosis, and belongs to the field of important epidemic prevention and cure. The immunosensor is a novel biosensor which is constructed through combining an immunoassay and a high sensitive sensing technology, and is applicable for analyzing and studying trace quantity of immunogenicity substance. According to the present invention, based on preparatory antigen marking and screening work, mycobacterium tuberculosis Rv2175c gene encoding protein (hereinafter referred to as specific antigen) is adopted as a specific antigen, a high sensitive electrochemical immunosensor is constructed, an antibody recognized by the specific antigen is detected in serum to realize the tuberculosis serodiagnosis. A method for the tuberculosis serodiagnosis comprises the following steps: (1) cloning the Rv2175c gene of the mycobacterium tuberculosis H37Rv; (2) heterologous expressing the purified specific antigen in escherichia coli; (3) preparing antibody serum on rabbits by using the specific antigen to be adopted as a positive control; (4) preparing the electrochemical immunosensor; (5) detecting the serum of healthy people and the serum of tuberculosis patients. With the present invention, the healthy people and the tuberculosis patients can be distinguished well through difference of current changing detected by the immunosensor, such that a purpose of serodiagnosis is achieved.

Description

technical field [0001] The technology relates to the fields of specific electrochemical immunosensor and immunological diagnosis of tuberculosis. Background technique [0002] Tuberculosis is an important chronic infectious disease that seriously threatens human health. According to the latest research report released by WHO: In 2008, there were 9.4 million new tuberculosis infections worldwide (15% of which were HIV positive), 11.1 million tuberculosis patients, and 1.1 million deaths due to tuberculosis. If this trend continues, by 2015, the number of new tuberculosis infections worldwide will reach 10 million. The co-infection of Mycobacterium tuberculosis and HIV, the emergence and spread of multidrug-resistant and broad-spectrum drug-resistant tuberculosis have further stimulated the task of global tuberculosis prevention and control. [0003] Diagnosis is the premise of treatment, but the current diagnostic techniques have greatly hampered the control of tuberculosis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N27/26C12N15/31C12N15/70C07K14/35
Inventor 谢建平周培富袁若袁亚利
Owner SOUTHWEST UNIVERSITY
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