Single strand nucleic acid detection kit and method as well as application of single strand nucleic acid detection kit and method

A detection kit, single-stranded nucleic acid technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of increasing samples, insufficient signal strength, low signal amplification efficiency, etc., to reduce the possibility of contamination , The effect of improving signal amplification efficiency and high practical application value

Active Publication Date: 2018-02-06
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Although the target sequence of this technology can be a single-stranded oligonucleotide, because it is a single-cycle linear signal amplification technology, the signal amplification efficiency is low, and the signal intensity generated within a certain period of time is insufficient, resulting in low detection sensitivity.
In addition, for some double-cycle or multi-cycle signal amplification techniques, because elements in different cycles interfere with each other, multi-step operations are required to achieve signal amplification, which increases the complexity of the operation and increases the possibility of sample contamination.

Method used

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  • Single strand nucleic acid detection kit and method as well as application of single strand nucleic acid detection kit and method
  • Single strand nucleic acid detection kit and method as well as application of single strand nucleic acid detection kit and method
  • Single strand nucleic acid detection kit and method as well as application of single strand nucleic acid detection kit and method

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preparation example Construction

[0045] Preferably, in the above-mentioned single-stranded nucleic acid detection kit, the preparation method of the circular deoxyribozyme comprises:

[0046] The linear deoxyribozyme-complementary nucleic acid fragment oligonucleotide single strand phosphorylated at the 5' end is connected into a circle under the action of DNA ligase;

[0047] Preferably, when linking into a circle under the action of DNA ligase, short-stranded DNA is used as a template for ligation, or an enzyme that ligates single-stranded DNA is used for direct ligation; more preferably, the double-stranded parts at both ends of the junction are respectively more than 5 bp. The schematic diagram of the preparation principle of circular deoxyribozyme is as follows: figure 2 shown.

[0048] Preferably, the DNA ligase includes T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, TaqDNA ligase, E.Coli DNA ligase, 9 ° NTM DNA ligase, CircLigase TM ssDNA Ligase, CircLigase TM Any of ssDNA Ligase II.

[0049] Prefer...

Embodiment 1

[0057] 1. Preparation and purification of circular deoxyribozymes

[0058] Add 1 μM oligonucleotide single-strand (containing deoxyribozyme sequence) modified by phosphorylation at the 5’ end to the ring-forming system, 2 μM DNA template strand and 5U T4 DNA Ligase (purchased from Thermo Scientific Company), and add 1× T4 DNALigase Buffer (containing 40mM Tris-HCl, 10mM MgCl 2 , 10mM DTT, 0.5mM ATP, pH 7.8@25℃), the total system is 100μL. After reacting at a constant temperature of 20°C for 12 hours in a PCR instrument, the enzyme was inactivated by incubating at 65°C for 10 minutes.

[0059] After concentrating the cyclized product to about 10 μM, use 15% denaturing polyacrylamide gel for gel cutting recovery, alcohol precipitation and cutting gel recovery product, after dissolving in sterilized water, use Nanodrop2000 to measure the concentration of circular deoxyribozyme.

[0060] 2. Signal amplification reaction

[0061] Add 200nM molecular beacon (stem-loop structure, ...

Embodiment 2

[0068] 1. Preparation and purification of circular deoxyribozymes

[0069] Add 0.5 μM 5’ end phosphorylated modified oligonucleotide single strand (containing DNAzyme sequence) at 0.5 μM, 100 U CircLigase TM ssDNA Ligase II (purchased from Epicenter), 1×CircLigase II Reaction Buffer (containing 33mM Tris-Ac, 66mM KAc, 0.5mM DTT, pH 7.5), 2.5mM MnCl 2 and 1M betaine, the total system is 100 μL. After reacting at a constant temperature of 60°C for 16 hours in a PCR instrument, the enzyme was inactivated by incubating at 80°C for 10 minutes.

[0070] Add 2 μL Exonulease I (purchased from Thermo Scientific) and 1×Reaction Buffer (containing 67mM glycine-KOH, 6.7mM MgCl 2 , 1 mM DTT, pH 7.5), after a constant temperature reaction at 37 ° C for 2 h, the enzyme was inactivated by incubating at 80 ° C for 15 min. The cyclized product after enzymatic digestion is purified by alcohol precipitation to remove impurities such as mononucleotides and ions. Finally, 20 μL of sterilized wa...

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Abstract

The invention belongs to the technical field of molecular detection and particularly relates to a single strand nucleic acid detection kit and method as well as application of the single strand nucleic acid detection kit and method. The shown kit comprises cyclic dnazyme and a molecular beacon; the cyclic dnazyme has a dnazyme sequence. The kit can perform double cycle amplification on a fluorescence signal of the molecular beacon through simple and convenient operation of a one-step method under the assistance of RNase H so as to realize high-sensitivity specific detection on the single strand nucleic acid and is finally applied to detection of the single strand nucleic acid such as viruses or miRNA.

Description

technical field [0001] The invention relates to the technical field of molecular detection, in particular to a single-stranded nucleic acid detection kit, method and application thereof. Background technique [0002] Nucleic acid detection methods have the advantages of high sensitivity, strong specificity and good stability, so they have been widely used in various research fields. Among them, nucleic acid signal amplification technology is a kind of detection technology that uses nucleic acid components to achieve optical or electrochemical signal enhancement in the system through amplification cycles. Nucleic acid signal amplification technology can realize highly sensitive detection of nucleic acid molecules, proteins, small molecular substances or ions. Therefore, it has been widely used in many fields such as food science, molecular biology and medicine in recent years. [0003] At present, researchers have developed a variety of nucleic acid signal amplification tech...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682C12Q1/70
CPCC12Q1/682C12Q1/70C12Q2521/345
Inventor 安然梁兴国
Owner OCEAN UNIV OF CHINA
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