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Real-time fluorescence constant temperature index amplification method

A constant-temperature exponential amplification and real-time fluorescence technology, applied in the fields of life science and biology, can solve problems such as limiting the application value of constant-temperature amplification technology, and achieve the effect of ensuring specificity

Inactive Publication Date: 2015-12-16
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned technical bottlenecks limit the application value of the existing constant temperature amplification technology to a certain extent, especially in the field of life sciences where multiple detection of target molecules is required as much as possible, such as the detection of infectious pathogens and miRNA

Method used

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Examples

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Effect test

Embodiment 1

[0260] Example 1: Detection of oligonucleotide molecules

[0261] 1. Target-specific oligonucleotides (ODNs) TS ) and signal amplification template (T SA ) design and synthesis:

[0262] Design and synthesize target molecule-specific oligonucleotides (ODN) based on miRNA and other naturally occurring small fragment nucleic acid molecules with 3'-OH TS ) and signal amplification template (T SA ), wherein, SEQNo.1 is a miRNA mimic molecule, which has a 3'-OH that can trigger its own extension, and the nucleotide sequence composition characteristics from the 3'-end to the 5'-end of SEQNo.2 are as follows: ①with ODN TS The nucleotide sequence of the first region complementary to the nucleotide sequence; ②The NERS antisense strand sequence region of Nb.BsrDI nick endonuclease, that is, 5'-GCAATG-3'; ③The first region with the same nucleotide sequence as the first region The nucleotide sequence of the second region, and the fluorescent quenching group BHQ2 is labeled at the corr...

Embodiment 2

[0273] Embodiment two: the detection of genomic DNA

[0274] 1. Target-specific oligonucleotides (ODNs) TS ) preparation

[0275] According to the sequence characteristics (SEQNo.3) of the target molecule, it is designed to prepare the target molecule specific oligonucleotide (ODN TS ) signal generation template (T SP ) oligonucleotide (SEQNo.4), wherein the target molecule has the NERS sense strand sequence region of Nt.BstNBI nick endonuclease, ie 5'-GAGTC-3'. The total linear constant temperature amplification system is 20 μl, and the system includes the following components: 1× ReactionBuffer(NEB), 0.5×NEBuffer3.1(NEB), 0.4units / μlNt.BstNBINickingEnzyme(NEB), 0.05units / μl (exo-)DNA Polymerase (NEB), 2×EvaGreen (Biotium), 400 μM dNTPs (Promega), 10 μg / ml BSA (NEB), 100 nM SEQ No. 2, series final concentration SEQ No. 3. The reaction conditions are: 60°C for 40 minutes, and the fluorescence acquisition is once per minute; 80°C for 20 minutes to inactivate the nicking ...

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Abstract

The invention provides a real-time fluorescence constant temperature index amplification method. According to the method, an oligonucleotide signal amplification template of a stem-ring-tail structure and marked with fluorescence or an oligonucleotide signal amplification template of a pure linear structure and marked with fluorescence are used, the process of cutting, extending and chain replacing can be executed repeatedly at the constant temperature under the combined action of a nicking agent with one chain of a cut double-chain nucleic acid molecule and a DNA polymerase with chain replacing activity, so that target molecule specific oligonucleotides are amplified in an index form, and fluorescence signals can be released. By means of varieties and abundance of the fluorescence signals released by the signal amplification templates corresponding to the different target molecule specific oligonucleotides, multiple target molecules can be detected in parallel in a single reaction tube, a detection tank or a detection hole, the target molecule specific oligonucleotides can be amplified by 106 times or more, and the technical problem that by means of an existing constant temperature amplification technology, multiple target molecules cannot be detected at the same time in one detecting system can be effectively solved.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and is a real-time fluorescence constant temperature exponential amplification method that can simultaneously qualitatively and / or quantitatively detect multiple nucleic acid target molecules, and can be widely used in miRNA, mRNA, cDNA, genomic DNA and other various On-site rapid detection of target molecules. Background technique [0002] Nucleic acid amplification technologies (nucleic acid amplification technologies, NAT) are widely used in various fields of life sciences, such as genetic diseases, infectious diseases, tumor diseases, food safety, forensic science. Simplicity, efficiency, specificity, sensitivity, accuracy, precision, and affordability of nucleic acid amplification techniques are critical. [0003] At present, there are two main types of nucleic acid amplification technologies, namely variable temperature amplification technologies based on temperature thermal c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2561/113C12Q2561/101C12Q2531/113
Inventor 黄庆府伟灵
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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