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Method for detecting nucleic acid

A detection method and nucleic acid technology, applied in biochemical equipment and methods, microorganism determination/inspection, etc., can solve problems such as complicated operation, and achieve the effect of simple experimental device, convenient operation, and low professional technical requirements.

Active Publication Date: 2012-12-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention provides a nucleic acid detection method, which solves the problem that the traditional method finally needs to identify the target molecule through a detection instrument, and the operation is complicated

Method used

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  • Method for detecting nucleic acid
  • Method for detecting nucleic acid
  • Method for detecting nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Ligase Selection

[0040] Target molecule: ACTCAGAGGAAGAAAACGATGAAATAGATGGAG.

[0041] 1. Design the following padlocks and amplification primers according to the above target molecules.

[0042] Padlock probes (with a phosphate group at the 5' end):

[0043] PO 3 -GTTTTTCTTCCTCGCTAAGTCTAAGAAAGTAGGATAGGACAGATAGCCATCTATTTCATC.

[0044] Primer: TTTCTTAGACTTAGCG.

[0045] 2. Add the padlock probe, target nucleic acid molecule, ligase and reaction buffer to 10 μL of the ligation system, and react at the corresponding temperature for 1 hour.

[0046] In this example, three kinds of ligases were used respectively, and the formulation of the connection system and the conditions of the connection reaction are as follows:

[0047] 1) Taq DNA ligase

[0048] 10μL ligation system containing 20mM Tris-HCl (pH 7.6), 25mM KAc, 10mMMg(Ac) 2 , 10mM DTT, 1mM NAD, 0.1% Triton X-100, 20U thermophilicTaq DNA ligase (New England BioLabs, Jitai Bio, China), 500nM phosphoryla...

Embodiment 2

[0058] The ligation efficiency and sequence recognition ability of DNA ligase directly affect the sensitivity and specificity of the detection method. The ligation efficiency of T4 DNA ligase is relatively high, and the cost is low, but because its reaction temperature is only 16°C, at this temperature Under certain conditions, incompletely matched sequences (such as mutant sequences) tend to hybridize with padlock probes to form double strands. If the mutation site happens not to be at the ligation site, it will also be ligated, resulting in non-specific ligation and high background . The reaction temperature of Taq DNA ligase is relatively high (higher than 45°C), which is higher than the hybridization Tm of the mutant sequence. Under this condition, the mutant sequence will not hybridize with the padlock probe to form a double strand, so the background is relatively low. Example 2 Rolling Circle Amplification Time Optimization

[0059] Target molecule: ACTCAGAGGAAGAAAACGAT...

Embodiment 3

[0072] Embodiment 3 sensitivity analysis

[0073] The operation method of this embodiment is the same as that of Example 2, and the reaction time is selected as 4 hours, the difference is that the copy number of the target molecule is 10 respectively. 6 ~10 10 , the result is as Figure 4 As shown, it can be seen that this method can detect at least 10 7 Copies (equivalent to 17 amol) of the target molecule. In addition, it was also found that the copy number of the target molecule is within a certain range, and there is a linear relationship with the aggregation amount of the magnetic beads, and there are obvious differences in the aggregation and dispersion results of the magnetic beads, which is very easy to judge.

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Abstract

The invention discloses a method for detecting nucleic acid, which comprises the following steps that: (1) a lock type probe and an amplification primer are designed and synthesized according to the sequence of a target nucleic acid molecule; (2) a DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) reverse transcription product is treated in advance, and then is mixed with the lock type probe, ligase and buffer solution, and heating is carried out to inactivate the ligase after the ligation; (3) a ligation product is taken as a template, and the amplification primer is utilized for roll loop amplification; (4) after the roll loop amplification is completed, magnetic beads are added into a roll loop amplification system to gather the amplification products, and the whole material is transferred to filter paper to be aired; and (5) the surface of the filter paper is observed by naked eyes to judge whether the DNA or RNA contains the target nucleic acid molecule or not. The method requires simple experimental devices, only needs a pipettor and a constant temperature heater to complete the whole detection process, and is very low in cost compared with the conventional method.

Description

technical field [0001] The invention relates to a biomolecular detection technology, in particular to a nucleic acid detection method. Background technique [0002] Molecular diagnostic technology based on nucleic acid detection is a new disease diagnostic technology in addition to hematology, pathology, immunology and microbiology. Treatment provides information and a basis for decision-making. Real-time quantitative (reverse transcription) polymerase chain reaction (hereinafter referred to as qPCR or qRT-PCR) technology is one of the most widely used nucleic acid detection technologies. The technology is deficient in terms of sensitivity and detection time. [0003] However, the current commercialized qPCR instruments usually include two important components, a cycle temperature control system and a fluorescence detection system, resulting in a bulky, expensive, and inconvenient to carry and move the instrument. Moreover, most qPCR and qRT-PCR technologies use fluoresce...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 姚波林彩琴
Owner ZHEJIANG UNIV
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