Oligonucleotides aptamer of targeted mycobacterium tuberculosis Ag85B, preparation method and application thereof

Abstract

The invention discloses an oligonucleotides aptamer of targeted mycobacterium tuberculosis Ag85B, a preparation method and application thereof. The oligonucleotides aptamer has nucleotide sequences shown as AP-1 to AP-13. The preparation method of the oligonucleotides aptamer comprises steps as follows: (1) constructing a random single-stranded DNA (ssDNA) library and preparing a primer; (2) preparing the random single-stranded DNA (ssDNA) library by PCR amplification; (3) carrying out SELEX screening; (4) detecting the appetency; (5) cloning and sequencing DNA. The oligonucleotides aptamer of targeted mycobacterium tuberculosis Ag85B is applied to detect the antigen level of the mycobacterium tuberculosis Ag85B in human serum, can replace antibodies, improves the sensitivity and the specificity of the tuberculosis antigen detection in specimens of body fluid (such as serum, hydrothorax, cerebrospinal fluid ascites and the like), thereby improving the accuracy rate of tuberculosis diagnosis.

Description

technical field [0001] The invention relates to an oligonucleotide aptamer targeting Mycobacterium tuberculosis Ag85B and its preparation method and application, belonging to the technical field of tuberculosis medical immunology and detection. Background technique [0002] The tuberculosis epidemic and drug resistance in my country are quite serious, and the number of tuberculosis patients ranks second in the world, second only to India. There are 4.51 million pulmonary tuberculosis patients, including 1.96 million infectious pulmonary tuberculosis patients, 1.4 million new tuberculosis cases and 130,000 annual deaths, ranking first among infectious diseases. Among the diagnostic methods routinely used in clinical practice at present, the sensitivity of clinical specimen smear detection is low, and the positive rate is only 20-30%; the positive rate of traditional Roche culture is low, only about 30%, and it takes 4-8 weeks. The serological diagnosis of tuberculosis is mai...

AI Technical Summary

Problems solved by technology

Among the diagnostic methods routinely used in clinical practice at present, the sensitivity of clinical specimen smear detection is low, and the positive rate is only 20-30%; the positive rate of traditional Roche culture is low, only about 30%, and it takes 4-8 weeks

The serological diagnosis of tuberculosis is mainly the detection of tuberculosis antibodies and antigens, and the detection of anti-tuberculosis antibodies is only an auxiliary diagnostic method. However, the detection sensitivity of tuberculosis antigen in body fluid samples is still low, and the main problems are as follows: ① Lack of high-titer specific antibodies. Although foreign countries have used specific tuberculosis antigens to immunize animals in recent years, and purified highly effective The research reports on antibodies are basically small sample size studies, lacking clinical evaluation of large sample size; There are certain limitations in the selection of target-specific antigens; ③The amount of Mycobacterium tuberculosis antigens in serum is low. Antigens combine with corresponding antibodies in vivo to form circulating immune complexes (CIC), which limits the sensitivity of antigen detection in serum

But so far, there are no reports about the Mycobacterium tuberculosis secreted antigen Ag85B aptamer and the application of oligonucleotide aptamer to detect tuberculosis antigen at home and abroad

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