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Method for detecting nucleic acid using asymmetric isothermal amplification of nucleic acid and signal probe

A detection method and isothermal amplification technology, applied in the field of needles, can solve problems such as false positive results and limitations, and achieve the effects of rapid and accurate amplification, accurate diagnosis, and no risk of contamination

Inactive Publication Date: 2016-09-21
DXGENE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] According to the inventor's prior patent (Korean Patent No. 10-0957057), a method of isothermally amplifying nucleic acid and signal probe simultaneously using the same amount of forward and reverse DNA-RNA-DNA hybrid primers and detecting the target nucleic acid The problem with the symmetric isothermal amplification method is that the sensitivity is 100 cells per reaction, which has limitations when used for diagnostics, and can also cause false positive results due to non-specific reactions

Method used

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  • Method for detecting nucleic acid using asymmetric isothermal amplification of nucleic acid and signal probe
  • Method for detecting nucleic acid using asymmetric isothermal amplification of nucleic acid and signal probe
  • Method for detecting nucleic acid using asymmetric isothermal amplification of nucleic acid and signal probe

Examples

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Effect test

Embodiment 1

[0066] Example 1: Isothermal amplification of tuberculosis DNA

[0067] As the target DNA, commercially available Mycobacterium tuberculosis DNA group DNA was quantitatively diluted and used as a template for the isothermal amplification reaction. (Vircell, Spain)

[0068] The external primers (SEQ ID NO: 1 and SEQ ID NO: 2) were designed to include a sequence complementary to the nucleotide sequence of Mycobacterium tuberculosis IS6110, and SEQ ID NO: 1 (forward, sense) and SEQ ID NO: 2 (reverse, anti-sense) are as follows.

[0069] Serial number 1: 5'-CGATCGAGCAAGCCA-3'

[0070] Serial number 2: 5'-CGAGCCGCTCGCTGA-3'

[0071] In the DNA-RNA-DNA hybrid primers (SEQ ID NO: 3 and SEQ ID NO: 4), the oligomeric DNA-RNA part at the 5' end has a sequence non-complementary to the base sequence of Mycobacterium tuberculosis IS6110, and the oligomeric DNA part at the 3' end It has a sequence complementary to the nucleotide sequence of Mycobacterium tuberculosis IS6110, and the sequ...

Embodiment 2

[0079] Example 2: Isothermal amplification of Chlamydia DNA

[0080] As target DNA, Chlamydi Atrachomatis DNA group DNA was used. In order to extract the DNA group DNA of Chlamydia, in the Chlamydia strain (ATCC Cat.No.VR-887), use the G-spinTM DNA group DNA extraction kit (iNtRON Biotechnology, Cat.No.17121), after the DNA group DNA is extracted, Perform the amplification reaction. When extracting the DNA group DNA, centrifuge 500 μl of the bacterial suspension at 13,000 rpm for 1 minute and remove the supernatant, hybridize a small amount of 500 μl of phosphate buffer saline (PBS) (pH 7.2) for centrifugation and remove the supernatant, add 300 μl of G-buffer solution of RNase A and Proteinase K were used to suspend the cell pellet and stand at 65° C. for 15 minutes. Next, 250 µl of binding buffer (Binding buffer) was added to uniformly hybridize, and then the spin column was bound to the DNA. After adding 500 ml of washing buffer (washing buffer) A, it was centrifuged at ...

Embodiment 3

[0092] Example 3: Isothermal Amplification of Neisseria gonorrhoeae DNA

[0093] As the target DNA, NeisseriA gonorrhoease DNA group DNA was used. In order to extract DNA group DNA of Neisseria gonorrhoeae strain (ATCC Cat. No. 49226), use G-spinTM DNA group DNA extraction kit (iNtRON Biotechnology, Cat. No. 17121), after extracting DNA group DNA, perform amplification reaction. When extracting the DNA group DNA, centrifuge 500 μl of the bacterial suspension at 13,000 rpm for 1 minute and remove the supernatant, hybridize a small amount of 500 μl of phosphate buffer saline (PBS) (pH 7.2) for centrifugation and remove the supernatant, add 300 μl of G-buffer solution of RNase A and Proteinase K were used to suspend the cell pellet and stand at 65° C. for 15 minutes. Next, 250 µl of binding buffer (Binding buffer) was added to uniformly hybridize, and then the spin column was bound to the DNA. After adding 500 ml of washing buffer (washing buffer) A, it was centrifuged at 1300...

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Abstract

The present invention relates to a method for accurately detecting a target nucleic acid by asymmetrically and isothermally amplifying the target nucleic acid using an external primer set, an internal primer set having different percentages of forward and reverse DNA-RAN-DNA hybrid primers, and a DNA-RNA-DNA hybrid signal probe and amplifying a signal of the probe at the same time. According to the present invention, the signal of the probe can be efficiently amplified compared with the method of the prior art, a symmetric iTPA method, which is an isothermal primer and probe amplification method using the same percentage of primers. Therefore, the present invention is applied to the accurate detection and confirmation of pathogens, detection of gene modification inducing identified phenotypes, diagnosis of susceptibility to genetic diseases, evaluation of gene expression, and various genome projects, and thus is useful in the molecular biological researches and disease diagnosis.

Description

technical field [0001] The present invention relates to the isothermal amplification method of nucleic acid and signal probe and the nucleic acid detection method utilizing the amplified signal probe, more specifically relates to the use of external primer pair, deoxyribonucleic acid-ribonucleic acid-deoxyribonucleic acid (DNA-RNA-deoxyribonucleic acid) DNA) hybridization primer pair and DNA-RNA-DNA hybridization signal probe, amplify target nucleic acid and signal probe simultaneously, and the method for rapidly detecting target nucleic acid. It is further specifically related to the use of different ratios of forward DNA-RNA-DNA hybrid primers and reverse DNA-RNA-DNA hybrid primers to amplify nucleic acids in an asymmetric manner, and at the same time, utilize The DNA-RNA-DNA hybridization signal probe of the base sequence complementary to the DNA asymmetrically over-amplified due to the excessive use of primers in the DNA and amplifies the signal, thereby detecting nucleic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q1/6853C12Q2521/327C12Q2525/121C12Q2531/107C12Q2531/119C12Q2565/1015C12Q1/6832
Inventor 金珉煥
Owner DXGENE
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