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Loop-mediated isothermal amplification method implemented by combining antarctic thermal sensitive uracil deoxyribonucleic acid glycosylase (AUDG) with self-avoiding molecule recognition system (SAMRS)

A loop-mediated constant temperature and constant temperature amplification technology, applied in the field of microorganisms and molecular biology, can solve the problems of false positive results and cross-contamination, and achieve the effect of eliminating false positive results, increasing specificity and ensuring high efficiency.

Active Publication Date: 2018-01-09
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to apply the LAMP amplification product to the detection of biosensing technology, opening the reaction tube is a necessary step, which causes a large amount of amplification product to volatilize in the form of aerosol, resulting in cross-contamination and false positive results
In addition, due to the technical design of LAMP, this technology is prone to false positive results, which are mainly caused by hybridization between primers, self-assortment within primers or off-target hybridization.

Method used

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  • Loop-mediated isothermal amplification method implemented by combining antarctic thermal sensitive uracil deoxyribonucleic acid glycosylase (AUDG) with self-avoiding molecule recognition system (SAMRS)
  • Loop-mediated isothermal amplification method implemented by combining antarctic thermal sensitive uracil deoxyribonucleic acid glycosylase (AUDG) with self-avoiding molecule recognition system (SAMRS)
  • Loop-mediated isothermal amplification method implemented by combining antarctic thermal sensitive uracil deoxyribonucleic acid glycosylase (AUDG) with self-avoiding molecule recognition system (SAMRS)

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Experimental program
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Effect test

Embodiment 1

[0057] Example 1. LAMP reaction amplification

[0058] 1. Principle of LAMP reaction

[0059] The LAMP reaction system includes 6 primers, which recognize 8 regions of the target sequence, including 2 inner primers, namely inner primers FIP and BIP; 2 replacement primers, namely F3 and B3; 2 loop primers, namely LF and LB. In order to construct a detectable product, any one of the 6 primers can be selected and labeled with a hapten (Dig, Digoxigenin) at the 5' end, and the newly labeled primers are named F3*, B3*, FIP*, BIP*, LF* and LB*. In the present invention, LF* is taken as an example to illustrate the principle of the present invention.

[0060] Under the given constant temperature conditions, the double-stranded DNA in the reaction system is in a dynamic equilibrium state of half-dissociation and half-binding. When any primer performs base pairing extension to the complementary part of the double-stranded DNA, the other strand will be dissociated. From, become a sin...

Embodiment 2

[0071] Embodiment 2.SAMRS-LAMP reaction system

[0072] 1. SAMRS-LAMP reaction principle

[0073] The reaction principle of SAMRS-LAMP is the same as that of ordinary LAMP reaction. Only the SAMRS components are used to modify the ordinary LAMP primers, which enhances the specificity of the primers, thereby enhancing the specificity of the method, thus eliminating the problems caused by off-target hybridization and primer dimers. False positive results.

[0074] 2. SAMRS-LAMP reaction system:

[0075] The concentration of SAMRS-FIP is 40pmol, the concentration of SAMRS-BIP is 40pmol, the concentration of SAMRS-F3 and SAMRS-B3 is 10pmol, the concentration of SAMRS-LF* and SAMRS-LB is 20pmol, 2M Betain, 8mM MgSO 4 , 2.5 μL of 10×Bst DNA polymerase buffer, 1.4 mM dATP, 0.7 mM dTTP, 0.7 mM dUTP, 1.38 mM dCTP, 0.02 mM biotin-14-dCTP, 1.4 mM dGTP, 10 U of chain Replace DNA polymerase, 1 μL template, add deionized water to 25 μl. The entire reaction was kept at 60°C for 1.5 hours...

Embodiment 3

[0084] Embodiment 3.AUDG-SAMRS-LAMP reaction system:

[0085] The concentration of SAMRS-FIP is 40pmol, the concentration of SAMRS-BIP is 40pmol, the concentration of SAMRS-F3 and SAMRS-B3 is 10pmol, the concentration of SAMRS-LF* and SAMRS-LB is 20pmol, 2M Betain, 8mM MgSO 4, 2.5 μL of 10×Bst DNA polymerase buffer, 1.4 mM dATP, 0.7 mM dTTP, 0.7 mM dUTP, 1.38 mM dCTP, 0.02 mM biotin-14-dCTP, 1.4 mM dGTP, 10 U of chain Replace DNA polymerase, 1U Antarctic thermosensitive uracil deoxynucleic acid glycosylase, 1 μL template, add deionized water to 25 μl. The entire reaction was kept at 60°C for 1.5 hours, and the reaction was terminated at 80°C for 5 minutes.

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Abstract

The invention discloses a loop-mediated isothermal amplification method implemented by combining antarctic thermal sensitive uracil deoxyribonucleic acid glycosylase (AUDG) with a self-avoiding molecule recognition system (SAMRS). According to the method, in a process of loop-mediated isothermal amplification, SAMRS modification is performed on four bases from the reciprocal second base to the reciprocal fifth base at the 3' end of a primer, and the 5' end of a primer LF is labeled with a hapten; the AUDG, deoxyuridine and biotinylated deoxycytosine are introduced into an amplification system;the loop-mediated isothermal amplification method is based on a loop-mediated isothermal amplification technology and is used for detecting amplification products in combination of a polymer nanometer biosensor. The method aims at the fact that the amplification products of an IS6110 specific sequence of a mycobacterium tuberculosis complex group can be visually detected by the polymer nanometerbiosensor. The method is convenient and fast, rapid, sensitive and specific, and is suitable for detecting various nucleotide fragments.

Description

technical field [0001] The invention discloses a method for detecting microbial target genes through loop-mediated constant temperature amplification, which belongs to the technical field of microorganisms and molecular biology. Background technique [0002] In the fields of modern biology and medicine, nucleic acid amplification is an indispensable technology, which has been widely used in basic research, clinical diagnosis, archaeological research, epidemiological research, transgenic research and other fields. Among the nucleic acid amplification technologies that have been developed, the polymerase chain reaction (Polymerase Chain Reaction, PCR) is the first established in vitro nucleic acid amplification technology, which has epoch-making significance. This technology has been widely used in biological related fields . However, when PCR technology is used for nucleic acid amplification, it is limited by laboratory conditions and relies on complex and expensive thermal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N33/53
Inventor 叶长芸王毅王艳
Owner ICDC CHINA CDC
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