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Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction

A technology of constant temperature amplification and polymerase, applied in biochemical equipment and methods, microbial measurement/testing, DNA preparation, etc., can solve the problems of difficult recovery and sequencing of LAMP products, unclonable LAMP products, complex LAMP products, etc., and achieve Suitable for large-scale promotion and application, shortened reaction time, and simple primer design

Active Publication Date: 2014-12-24
中国人民解放军疾病预防控制中心
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0016] The most advantageous of these constant temperature nucleic acid amplification methods is the LAMP method, but the LAMP product is too complex, and the recovery and sequencing of the LAMP product is very difficult, and it cannot be directly sequenced like ordinary PCR products; because the LAMP product is an extremely complex and irregular amplification amplification mixture, so LAMP products cannot be used for cloning
These shortcomings limit the application of LAMP to the rapid detection of genes

Method used

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  • Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction
  • Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction
  • Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction

Examples

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Effect test

Embodiment 1

[0087] Embodiment 1, use polymerase spiral reaction (PSR) to detect whether to contain NDM-1 gene in the sample to be tested

[0088] Taking the NDM-1 gene as an example, the polymerase helix reaction (PSR) is used to detect whether the target gene (DNA) is contained in the sample to be tested. The specific method includes the following steps:

[0089] 1. Design of PSR primers

[0090] The nucleotide sequence of the NDM-1 gene (GenBank number: FN396876) was retrieved from the American gene database GenBank, and homology analysis was performed by BLAST software to find the conserved target sequence (sequence 1 in the sequence table), and then based on its conserved target sequence Design PSR primers.

[0091] Same as ordinary PCR, first design a pair of primers F and B, F primer: 5'-GGTCGATACCGCCTGGAC-3', B primer: 5'-GCATGCAGCGCGTCCA-3', and then add a general foreign sequence at the 5' end of the two N, F primers add sequence N (5'-3': ACGATTCGTACATAGAAGTATAG) to form prime...

Embodiment 2

[0110] Embodiment 2, detect whether contain RNA in the sample to be tested with polymerase spiral reaction (PSR)

[0111] Taking the detection of H1N1 virus as an example, polymerase spiral reaction (PSR) is used to detect whether RNA is contained in the sample to be tested. The specific method includes the following steps:

[0112] 1. Design of PSR primers

[0113] The nucleotide sequence of the H1N1 gene (GenBank number: KM361419.1) was retrieved from the American gene database GenBank, and the homology analysis was performed by BLAST software to find the conserved target sequence (sequence 4 in the sequence table), and then according to its conserved target sequence Design PSR primers.

[0114] Same as ordinary PCR, first design a pair of primers F and B, F primer: 5'-GCAATGAGAACTATTGGGACTC-3', B primer: 5'-ATTTGCTGCAATGACGAGAG-3', and then add a general foreign sequence at the 5' end of the two N, F primers add sequence N (5'-3': ACGATTCGTACATAGAAGTATAG) to form primer F...

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Abstract

The invention discloses a nucleic acid isothermal amplification method by polymerase spiral reaction (PSR). A pair of oligonucleotide primers is used, mutually reverse nucleotide fragments are added to 5' ends of the primers, a target gene is subjected to PSR under the action of the primers and deoxyribonucleic acid (DNA) polymerase and under the isothermal condition, self-spiral extending of the target gene can be achieved, and thereby, nucleic acid amplification can be finished. According to the nucleic acid isothermal amplification method by PSR, a novel technology platform is provided for nucleic acid detection, amplification of a product is simple, an amplification product is applicable to the field of detection and can be slightly processed to be subjected to clone recycling and sequencing, the method can be applied to all fields which require nucleic acid amplification, the market prospect is wide, the economic and social benefits are large, and the method is suitable for large-range popularization and application.

Description

technical field [0001] The invention belongs to a constant temperature nucleic acid amplification method in the technical field of molecular biology, in particular to a method for constant temperature amplification of nucleic acid by polymerase helical reaction and its application in nucleic acid (DNA or RNA) detection. Background technique [0002] Polymerase chain reaction (polymerase chain reaction), that is, PCR technology, is a method for rapidly amplifying a specific gene or DNA sequence in vitro invented by K.B. Heat-resistant DNA polymerase mixes primers and target DNA, and undergoes three cycles of high-temperature denaturation, low-temperature annealing, and suitable temperature extension as a cycle, and a large amount of target DNA is amplified in a short time. PCR technology has outstanding advantages such as specificity, sensitivity, high yield, rapidity, simplicity, good repeatability, and easy automation; it can amplify the target gene or a certain DNA fragmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
CPCC12Q1/6844C12Q2525/125C12Q2525/307C12Q2527/101C12Q2535/125C12Q1/68C12Q1/6853C12Q1/686
Inventor 刘威袁静陈泽良黄留玉董德荣
Owner 中国人民解放军疾病预防控制中心
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