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PCR analyzing method for quantitatively detecting nucleic acid through RNA polymerase and ligase coupled reaction medium

A technology of RNA polymerase and analysis method, which is applied in the field of analysis and detection, can solve the problem of low sensitivity, and achieve the effect of good selectivity, simple operation process and strong specificity

Inactive Publication Date: 2014-09-10
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Aiming at the problem that the sensitivity of the existing ligase-mediated PCR method is not high, the object of the present invention is to provide a highly sensitive PCR analysis method for nucleic acid detection mediated by RNA polymerase and ligase coupling reaction

Method used

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  • PCR analyzing method for quantitatively detecting nucleic acid through RNA polymerase and ligase coupled reaction medium
  • PCR analyzing method for quantitatively detecting nucleic acid through RNA polymerase and ligase coupled reaction medium
  • PCR analyzing method for quantitatively detecting nucleic acid through RNA polymerase and ligase coupled reaction medium

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Embodiment 1

[0047] The analytical method for detecting the DNA target (5′-TTGAGGTGCATGTTTGTGCC-3′) by PCR mediated by T7 RNA polymerase and T4 DNA ligase coupling reaction, the detection principle is as follows figure 1 shown. The sequence complementary to the DNA target in probe 1 (5′-GGCACAAACAT-3′), the sequence complementary to the DNA target in probe 2 (5′-GCACCTCAA-3′) and the T7 promoter sequence (5′-CCCTATAGTGAGTCGTATTAGTGATC- 3').

[0048] The following specific implementation of DNA target detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The specific operation steps are as follows:

[0049] First prepare the Part A ligation reaction solution. Add 1 μL of transcription buffer solution (20 mM Tris-HCl, 3 mM MgCl 2 , 5mM DTT, 5mM NaCl and 1mM spermidine, pH 7.9), 1μL probe 1 solution (5nM), 1μL probe 2 so...

Embodiment 2

[0057] The analytical method for detecting miR-122 (5′-UGGAGUGUGACAAUGGUGUUUG-3′) by PCR mediated by T7 RNA polymerase and T4 RNA ligase 2 coupling reaction, the detection principle is as follows Figure 4 shown. Probe 1 sequence (5′-GGTATCCAGGGAAGTGGATACGAAGAATGCACAAACACCATTG-3′), probe 2 sequence (5′-(P)TCACACTCCACGCTGTGCCCTATAGTGAGTCGTATTAGTGATC-3′), forward primer sequence (5′-GGGAAGTGGATACGAAGAATGC-3′), reverse primer sequence ( 5'-GATCACTAATACGACTCACTATAGG-3').

[0058] The following specific implementation of target miR-122 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The specific operation steps are as follows:

[0059] First prepare the Part A ligation reaction solution. Add 1 μL of transcription buffer solution (20 mM Tris-HCl, 3 mM MgCl 2, 5mM DTT, 5mM NaCl and 1mM spermidine, pH 7.9),...

Embodiment 3

[0066] The analytical method for detecting the DNA target (5'-TATTTGCCCCATTTTT-3') from respiratory syncytial virus (RSV) by PCR mediated by SP6 RNA polymerase and T4 DNA ligase coupling reaction, the detection principle is as follows Figure 5 shown. Probe 1 sequence (5′-GGTATCCAGGGAAGTGGATACGAAGCGTCGATAAAAATGG-3′), probe 2 sequence (5′-(P)GGCAAATACCTTTGCTTCGCTTCTATAGTGTCACCTAAAT-3′), forward primer sequence (5′-ATTTAGGTGACACTATAGAAGCG-3′), reverse primer sequence ( 5'-AGGGAAGTGGATACGAAGC-3').

[0067] The following specific implementation of DNA target detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The specific operation steps are as follows:

[0068] First prepare the Part A ligation reaction solution. Add 1 μL of transcription buffer solution (20 mM Tris-HCl, 3 mM MgCl 2 , 5mM DTT, 5mM NaCl and...

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Abstract

The invention discloses a PCR analyzing method for quantitatively detecting nucleic acid through RNA polymerase and a ligase coupled reaction medium. The method comprises the following steps that when target nucleic acid exits, 3' end hydroxyl radical and 5' end phosphate group among ligase medium oligonucleotide probes are reacted to form phosphate diester bond, the coupling product has the RNA polymerase promoter sequence, the RNA polymerase is started to synthesize a large number of RNA fragments with the target nucleotide sequence, the RNA fragments can serve as the target nucleic acid to continue to mediate connecting reaction among the oligonucleotide probes, the connecting reaction efficiency is improved through the introduction of the RNA polymerase, more amplification templates are provided for subsequent PCR reaction, the strength of fluorescence signals in a reaction system is measured in real time, and the concentration of the target nucleic acid is worked out through the comparison with a standard work curve. The method has the advantages of being high in sensitivity and specificity, easy to design, short in reaction time, capable of being widely applied to the basic biology study and the nucleic acid detection in the clinical diagnosis.

Description

technical field [0001] The invention belongs to the field of analysis and detection, in particular to a PCR analysis method for quantitative detection of nucleic acid mediated by RNA polymerase and ligase coupling reaction. Background technique [0002] The detection of specific sequence nucleic acids is very important in modern biology and medicine. There are many current uses for the identification of specific DNA and RNA sequences, including personal identification in forensic science, gene and genome sequencing in basic biology and applied medicine, microbial identification in food and environmental samples, pathogen identification in human patients, cancer and antimicrobial therapy. Drug diagnosis, genetic prediction in disease progression and response to drug therapy, etc. Currently, nucleic acid detection methods mainly include polymerase chain reaction (PCR), nucleic acid sequence-dependent amplification (NASBA), loop-mediated isothermal amplification (LAMP), strand...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851
Inventor 叶邦策尹斌成于翠媛
Owner EAST CHINA UNIV OF SCI & TECH
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