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Multiple cross isothermal amplification method implemented by aid of AUDG [Antarctic thermal sensitive uracil-DNA (deoxyribonucleic acid)-glycosylase] coupled with self-avoiding molecular recognition systems

A technology of cross constant temperature amplification and constant temperature amplification, applied in biochemical equipment and methods, analytical materials, recombinant DNA technology, etc., can solve the problems of false positive results, cross contamination, etc., to eliminate false positive results, increase specificity performance, ensuring high efficiency

Active Publication Date: 2017-12-29
ICDC CHINA CDC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to apply the MCDA 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

Method used

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  • Multiple cross isothermal amplification method implemented by aid of AUDG [Antarctic thermal sensitive uracil-DNA (deoxyribonucleic acid)-glycosylase] coupled with self-avoiding molecular recognition systems
  • Multiple cross isothermal amplification method implemented by aid of AUDG [Antarctic thermal sensitive uracil-DNA (deoxyribonucleic acid)-glycosylase] coupled with self-avoiding molecular recognition systems
  • Multiple cross isothermal amplification method implemented by aid of AUDG [Antarctic thermal sensitive uracil-DNA (deoxyribonucleic acid)-glycosylase] coupled with self-avoiding molecular recognition systems

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1.MCDA amplification

[0063] 1. MCDA reaction principle

[0064] The MCDA reaction system includes 10 primers, which recognize 10 regions of the target sequence, including 2 internal cross primers, namely CP1 and CP2 (Cross Primer, CP), 2 displacement primers (Displacement amplification), namely F1 and F2, and 6 Amplification primers, namely D1, C1, R1, D2, C2 and R2. In order to construct a detectable product, select any one of the 10 primers and label the hapten (FITC, fluorescein) at the 5' end, and the newly labeled primers are named F1*, F2*, CP1*, CP2*, C1* , C2*, D1*, D2*, R1* and R2*. In the present invention, C1* is taken as an example to illustrate the principle of the present invention.

[0065] 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 dou...

Embodiment 2

[0076] Embodiment 2.SAMRS-MCDA reaction system

[0077] 1. SAMS-MCDA reaction principle

[0078] The reaction principle of SAMS-MCDA is the same as that of ordinary MCDA reaction. Only the ordinary MCDA primers are modified with SAMRS components, 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.

[0079] 2. SAMS-MCDA reaction system

[0080] The concentration of the cross primer SAMRS-CP1 was 40pmol, the concentration of the cross primer SAMRS-CP2 was 40pmol, the concentration of the displacement primers SAMRS-F1 and SAMRS-F2 was 10pmol, and the amplification primers SAMRS-C1*, SAMRS-C2, SAMRS-R1, The concentration of SAMRS-R2, SAMRS-D1 and SAMRS-D2 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 cha...

Embodiment 3

[0087] The optimal reaction temperature determination of embodiment 3.SAMRS-MCDA technology

[0088] Under the conditions of the SAMRS-MCDA reaction system, the MTC template and the designed corresponding SAMRS-MCDA primers were added, and the template concentration was 2×10 3 copies / microliter (10pg / microliter). The reaction was carried out under constant temperature conditions (59-66°C), and the results were detected by a real-time turbidimeter, and different dynamic curves were obtained at different temperatures, see Figure 5 . 60-62°C is recommended as the optimum reaction temperature for SAMRS-MCDA primers. In the follow-up verification of the present invention, 61° C. was selected as the constant temperature condition for SAMRS-MCDA amplification.

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Abstract

The invention discloses a multiple cross isothermal amplification method implemented by the aid of Antarctic thermal sensitive uracil-DNA (deoxyribonucleic acid)-glycosylase (AUDG) coupled with self-avoiding molecular recognition systems (SAMRS). The multiple cross isothermal amplification method includes carrying out SAMRS modification on four basic groups from a second basic group to a fifth basic group from the bottom of a 3' end of each primer during multiple cross displacement amplification; labeling haptens at 5' ends of amplification primers C1 or C2; leading the AUDG, deoxidized uracil and biotinylated deoxidized cytosine into amplification systems; detecting amplification products by the aid of coupled macromolecule nano biosensing on the basis of multiple cross displacement amplification technologies. The multiple cross isothermal amplification method has the advantages that the amplification products of IS6110 specific sequences of mycobacterium tuberculosis complexes can be pertinently visually detected by macromolecule nano biosensors; the multiple cross isothermal amplification method is convenient, speedy, sensitive and specific and is suitable for detecting diversified nucleotide fragments.

Description

technical field [0001] The invention discloses a method for detecting microbial target genes through multi-cross displacement 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 expensi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N33/68G01N33/558
CPCC12Q1/6844G01N33/558G01N33/68C12Q2531/119
Inventor 叶长芸王毅王艳
Owner ICDC CHINA CDC
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