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Method for rapid fluorescence detection of polynucleotide target objects simultaneously at room temperature and constant temperature

A technology of isothermal and target objects at room temperature, applied in the field of molecular biology, can solve the problems of simultaneous detection, large dependence, and inaccurate PCR product quantification, etc., and achieve the goals of increasing detection accuracy, flexible and simplified selection, and avoiding damage Effect

Inactive Publication Date: 2015-10-14
ZHEJIANG SHANCE HEQISHI BIO SCI& TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a kind of method for the rapid simultaneous detection of multi-nucleic acid target objects of room temperature isothermal fluorescence in view of the above problems, to overcome the dependence of current fluorescent PCR on precision instruments, and the PCR product quantification of nucleic acid isothermal amplification technology is not accurate enough. It is not yet possible to detect multiple targets at the same time

Method used

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  • Method for rapid fluorescence detection of polynucleotide target objects simultaneously at room temperature and constant temperature
  • Method for rapid fluorescence detection of polynucleotide target objects simultaneously at room temperature and constant temperature
  • Method for rapid fluorescence detection of polynucleotide target objects simultaneously at room temperature and constant temperature

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Embodiment 1

[0052] This example is used to illustrate the dual fluorescence reaction at room temperature and constant temperature performed on the Twista instrument.

[0053] 1. According to NCBI GenBank and related literature reports, a 523bp sequence (SEQ ID No.6) was selected from the insertion sequence IS6110 of MTB for gene synthesis, and cloned into the vector pUC57 to prepare the recombinant plasmid pUC57-6110.

[0054] 2. Artificially synthesized a 141bp nucleotide sequence (SEQ ID No.7), which does not exist in nature, carried out gene synthesis, and cloned it into the vector pUC57 to prepare a recombinant plasmid pUC57-IC.

[0055] 3. Based on the design principles of primers and fluorescent probes, two pairs of primers were designed according to the sequences of SEQ ID No.6 and SEQ ID No.7, namely 6110-F / R (SEQ ID No.8 / 9) and IC-F / R (SEQ ID No.10 / 11); and two fluorescent probes, respectively Pro-6110 (SEQ ID No.12) and Pro-IC (SEQ ID No.13).

[0056] SEQ ID No. 6:

[0057] 5...

Embodiment 2

[0077] This example is used to illustrate the normal temperature and constant temperature triple fluorescence reaction performed on the ABI7500 instrument.

[0078] 1. According to NCBI GenBank and related literature reports, a 600bp sequence (SEQ ID No.14) was selected from the insertion sequence IS1081 of MTB for gene synthesis, and cloned into the vector pUC57 to prepare a recombinant plasmid pUC57-1081.

[0079] 2. According to NCBI GenBank and related literature reports, a 500bp sequence (SEQ ID No.15) was selected from the HBB gene in the human genome for gene synthesis, and cloned into the vector pUC57 to prepare a recombinant plasmid pUC57-HBB.

[0080] 3. Based on the design principles of primers and fluorescent probes, two pairs of primers were designed according to the sequences of SEQ ID No.9 and SEQ ID No.10, namely 1081-F / R (SEQ ID No.16 / 17) and HBB-F / R (SEQ ID No.18 / 19); and two fluorescent probes, respectively Pro-1081 (SEQ ID No.20) and Pro-HBB (SEQ ID No.21)...

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Abstract

The invention relates to a method for rapid fluorescence detection of polynucleotide target objects simultaneously at room temperature and constant temperature. The method comprises the steps that at the constant temperature, single-chain binding proteins partially open parental chains of double chains of multiple templates to form single chains; recombinases are bonded with multiple pairs of primers to form complexes which are bonded to the parental chains under the action of accessory proteins, and multiple fluorescence probes are also bonded with a complementation region; DNA polymerases are bonded to the 3' terminals of the primers so as to perform subchain extension; exonucleases recognize tetrahydrofuran sites on the multiple fluorescence probes which are under the double chain condition, so that fluorophores and quenching groups are separated after digestion, and fluorescence is released; after the multiple fluorescence probes are cut, 3'-OH ends which are originally closed due to modification of the 3' terminals of the probes are exposed, and the DNA polymerases can further extend to form subchains; to-be-tested samples can be subjected to qualitative and semi-quantitative determination through detecting the shape of amplification curves and the strength of fluorescence signals. The method disclosed by the invention can be used for qualitative and semi-quantitative determination of multiple target objects simultaneously at room temperature and constant temperature.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a method for rapidly and simultaneously detecting multiple nucleic acid targets under room temperature isothermal fluorescence, which is a method for simultaneously performing rapid, real-time and specific detection of multiple nucleic acid targets under normal temperature and isothermal conditions , and has broad application prospects in the fields of in vitro clinical testing, food safety, biosafety and agriculture. Background technique [0002] Nucleic acid isothermal amplification technology is a general term for a class of molecular biology techniques, which can be kept constant at a specific temperature to achieve a rapid increase in the copy number of a specific DNA or RNA fragment. In the field of nucleic acid amplification technology, nucleic acid non-isothermal amplification technology represented by polymerase chain reaction (PCR) has been used in the clinical diagnosis ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2521/319C12Q2522/101C12Q2525/186C12Q2537/143C12Q2563/107
Inventor 李贤祯周国辉程奇
Owner ZHEJIANG SHANCE HEQISHI BIO SCI& TECH CO LTD
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