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Hypersensitive molecular lock and key immune polymerase chain reaction detection method

A chain reaction and detection method technology, applied in the field of medical laboratory science, can solve the problems of contaminated negative samples, false positive results, etc., and achieve the effects of high sensitivity detection, sensitivity improvement, and high use value

Pending Publication Date: 2020-10-02
广州水石基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immuno-PCR method is not suitable for clinical use, because the open operation in the immune reaction will introduce a large amount of DNA aerosol, while the immuno-PCR method has extremely high sensitivity, and these aerosols will contaminate negative samples, resulting in a large number of false positives results appear

Method used

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  • Hypersensitive molecular lock and key immune polymerase chain reaction detection method
  • Hypersensitive molecular lock and key immune polymerase chain reaction detection method
  • Hypersensitive molecular lock and key immune polymerase chain reaction detection method

Examples

Experimental program
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Embodiment

[0032] Antigens of Mycobacterium tuberculosis were tested in blood samples, ascites, and cerebrospinal fluid using a hypersensitive molecular lock-key immunopolymerase chain reaction assay.

[0033] (1) Prepare experimental components and experimental equipment

[0034] (1) Components required for making experiments:

[0035] ① Preparation of recombinant Mycobacterium tuberculosis early secretion antigen target protein 6 (ESAT6) reference product, culture filtrate egg 10 (CFP10) reference product, PstS1 protein reference product: the genes of ESAT6 reference product, CFP10 reference product and PstS1 protein reference product The sequence was cloned into Escherichia coli expression vector PET28a;

[0036] Among them, the gene sequence of ESAT6:

[0037]ATGACAGAGCAGCAGTGGAATTTCGCGGGTATCGAGGCCGCGGCAAGCGCAATCCAGGGAAATGTCACGTCCATTCATTCCCTCCTTGACGAGGGGAAGCAGTCCCTGACCAAGCTCGCAGCGGCCTGGGGCGGTAGCGGTTCGGAGGCGTACCAGGGTGTCCAGCAAAAATGGGACGCCACGGCTACCGAGCTGAACAACGCGCTGCAGAACCTGGCGCGGACGA...

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Abstract

The invention discloses a hypersensitive molecular lock and key immune polymerase chain reaction detection method. The method comprises the following steps of (1) identifying target biological macromolecules by utilizing specific binding among biological macromolecules, wherein a sandwich compound is formed by the specific binding among the biological macromolecules, and a DNA fragment is connected to one monomer of the sandwich compound; (2) amplifying the DNA fragment connected with the sandwich compound by using a polymerase chain reaction to quantify target protein; and (3) eliminating theinterference of DNA aerosol on a test system by using molecular lock and key, so that the signal-to-noise ratio is improved by 3-4 orders of magnitude, and the sensitivity is also improved by 3 orders of magnitude. The invention belongs to the technical field of medical laboratory science, and particularly provides the hypersensitive molecular lock and key immune polymerase chain detection methodfor detecting protein target biomacromolecules in a medical sample with high sensitivity. The detection method has relatively high use value in the fields of diagnosis and monitoring of pathogens, tumors, neurodegenerative diseases, genetic diseases and the like.

Description

technical field [0001] The invention belongs to the technical field of medical laboratory science, and specifically refers to a hypersensitive molecular lock-and-key immunopolymerase chain reaction detection method. Background technique [0002] The detection of protein molecular markers is an important topic in medical testing. Common methods for detecting proteins include enzyme-linked immunoassay (ELSIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA) and immunofluorescence (IF), etc. In addition, mass spectrometry, flow cytometry, and protein chips can also be used for protein detection. The sensitivity of the above method can meet common application scenarios, but it is not suitable for some protein detection applications that require extremely high sensitivity. [0003] TakeshiSano etc. reported in 1992 that the use of immuno-PCR method can detect the number of 10 -23 Mole protein; Suman Sharma et al. reported in 2019 that the real-time immuno-PCR meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6804
CPCC12Q1/6804C12Q2531/113
Inventor 朱应竹谢正顺
Owner 广州水石基因科技有限公司