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A nucleic acid constant temperature amplification method initiated by a single primer

A constant-temperature amplification and single-primer technology, which is applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve the problems of high detection cost and complicated primer design

Active Publication Date: 2016-11-02
QINGDAO NAVID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In order to solve the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a nucleic acid constant temperature amplification method triggered by a single primer, which avoids the need to open the nucleic acid in advance by means of variable temperature in the existing traditional constant temperature detection method. The double-stranded nucleic acid is further amplified and the design of primers is complicated, and the detection cost is high.

Method used

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  • A nucleic acid constant temperature amplification method initiated by a single primer
  • A nucleic acid constant temperature amplification method initiated by a single primer
  • A nucleic acid constant temperature amplification method initiated by a single primer

Examples

Experimental program
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Effect test

Embodiment 1

[0078] Embodiment 1: Verify the feasibility of the method and the correctness of its principle.

[0079] In this embodiment, PBS plasmid is used as target nucleic acid, single primer is used for constant temperature detection of amplification, and the feasibility and correctness of principle of the method are verified by fluorescent signal. Add 1 μL (5×10 -6 M) molecular beacon MB1 (sequence is 5'-FAM-CGCTTGGTAGGCTCCGGTTCCCAACGATCAGATCCTGCTACCAAGCG-DABCYL-3', which is SEQ ID NO.1), 1×NEBuffer3.1 (100mM NaCl, 50mM Tris-HCl, 10mM MgCl 2 ,100μg / mL BSA pH7.9@25℃), 0.05μL Bst2.0WarmStart TM DNA polymerase, 0.4 μL Nt.BstNBI nicking enzyme, 1 μL (2.5 mM) dNTPs, 0.2 μL (10 μM) primer (the sequence is 5’-GGTTCCCAACGATCAGATCCTGGTGAGACTCAACTATGCTATTTCGTTCATC-3’, which is SEQ ID NO.2), 1 μL (1×10 -12 M) The target PBS plasmid (sequence is 5'-CTATTTCGTTCATCCATAGTTGCCTGACTC-3', which is SEQ ID NO.3), and finally add water to the system to 10 μL. Using Bole CFX96 TM The real-time fluore...

Embodiment 2

[0081] Example 2: Detection of PBS plasmids using a nucleic acid constant temperature amplification detection method initiated by a single primer.

[0082] In this example, different concentrations of PBS plasmids were detected to test the sensitivity of the nucleic acid detection method. Add 1 μL (5×10 -6 M) molecular beacon MB1 (ie, SEQ ID NO.1), 1×NEBuffer3.1, 0.05μL Bst2.0WarmStart TM DNA polymerase, 0.4 μL Nt.BstNBI nickase, 1 μL (2.5 mM) dNTPs, 0.2 μL (10 μM) primer (SEQ ID NO.2), and then add 1 μL 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 M, 1×10 -13 M, 1×10 -14 M, 1×10 -15 M, 1×10 -16 M and OM target PBS plasmids (ie, SEQ ID NO.3), add water to the system to 10 μL. Using Bole CFX96 TM The real-time fluorescent quantitative PCR instrument detects the fluorescent signal once per minute, and reacts at 55°C for 70 minutes; the results are as follows: Figure 4 As shown, the concentration gradient decreases from left to right. Figure 4 The result...

Embodiment 3

[0083] Example 3: Detection of the ability of the method to detect target nucleic acid against background interference in a complex system environment.

[0084] This example tests the anti-background interference ability of this scheme under the complex system containing the Escherichia coli genome. Add 1 μL (5×10 -6 M) molecular beacon MB1 (ie, SEQ ID NO.1), 1×NEBuffer3.1, 0.05μL Bst2.0WarmStart TM DNA polymerase, 0.4 μL nicking enzyme Nt.BstNBI, 1 μL (2.5 mM) dNTPs, 0.2 μL (10 μM) primer (ie, SEQ ID NO.2), 1 μL 1×10 -12 M PBS plasmid, and then add 1 μL 1×10 -10 M, 1×10 -11 M, 1×10 -12 M, 1×10 -13 M, 0M Escherichia coli genome, add water to the system to 10 μL at the end. Using Bole CFX96 TM The real-time fluorescent quantitative PCR instrument detects the fluorescent signal once every minute, and reacts at 55°C for 50 minutes. The detection result of this embodiment is as follows Figure 6 as shown, Figure 6 The middle curve results A, B, C, D, E are respectively...

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Abstract

The invention belongs to the technical field of nucleic acid detection, which concretely relates to a single primer-initiated nucleic acid constant temperature amplification method. The technical problem which need to be solved by the method is to provides the single primer-initiated nucleic acid constant temperature amplification method, the method avoids the disadvantages of complex primer design and high detection cost in a traditional constant temperature detection method in the prior art. The provided technical scheme is a single primer-initiated nucleic acid constant temperature amplification method, which is characterized in that the primers enable self complementation and pairing after pairing with nucleic acid by designing a primer sequence, then a stem-and-loop structure is formed, the primer can be disengaged from the target nucleic acid, and then nucleotide fragments can be continuously generated under combined action of cutting enzyme and polymerase. The technical scheme is completed under isothermal condition, the equipment capable of keeping constant temperature can be used for performing the method, and the operation is convenient; only one primer is required during the amplification process, so that detection scheme is simpler and easier to be carried out, and the nucleic acid amplification and detection efficiency can be increased.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a nucleic acid constant temperature amplification method initiated by a single primer. Background technique [0002] Nucleic acid detection has been widely used in many aspects such as clinical diagnosis, environmental monitoring, and prevention and control of infectious diseases. It has become the most widely used nucleic acid amplification method at present. However, traditional PCR requires the use of thermal denaturation methods to prepare single-stranded nucleic acid templates to anneal primers and templates. This process requires instruments with precise temperature control. In addition, the specificity of the PCR reaction depends on the specificity of the annealing of the primers. In order to obtain specific amplification, it is often necessary to optimize the annealing temperature, which is cumbersome. In addition, PCR products can also be used...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2521/101C12Q2521/301
Inventor 石超马翠萍韩典昂邓美莲
Owner QINGDAO NAVID BIOTECH CO LTD
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