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Loop-mediated isothermal amplification method based on taqman probe and its special lamp primer and kit

A loop-mediated constant temperature and probe technology, applied in the field of molecular biology detection of nucleic acids, can solve the problems of inability to amplify products specific detection, false positive detection results, non-specificity, etc. Simple operation and efficient amplification effect

Active Publication Date: 2015-10-28
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as long as nucleic acid is amplified by SYBR Green I dye, it will be positive, and it is non-specific; both hydroxynaphthol blue and calcein indicate the change of magnesium ions, a by-product of LAMP, and are also non-specific.
The third is the turbidity method. This method is mainly used to detect magnesium pyrophosphate, a by-product of LAMP. Magnesium pyrophosphate is a white precipitate. As long as the LAMP reaction solution occurs, the solution becomes turbid. This method is also non-specific.
[0011] Combining the above three detection methods, none of the amplification products can be specifically detected, and they are all indirect detection, which brings about a very big problem. If non-specific amplification occurs in LAMP, then the results obtained by these detection methods The results are all false positives. Therefore, it is necessary to improve the LAMP technology to fundamentally solve the problem of false positive test results.

Method used

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  • Loop-mediated isothermal amplification method based on taqman probe and its special lamp primer and kit
  • Loop-mediated isothermal amplification method based on taqman probe and its special lamp primer and kit
  • Loop-mediated isothermal amplification method based on taqman probe and its special lamp primer and kit

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Embodiment 1

[0101] Embodiment 1, detect NDM-1 gene with the LAMP method based on Taqman probe

[0102] Taking the NDM-1 gene as an example, the LAMP method based on the Taqman probe of the present invention is described in detail. The specific detection method includes the following steps:

[0103] 1. Primer design for the detection of NDM-1 gene based on Taqman probe for LAMP

[0104] The NDM-1 gene sequence (GenBank number: FN396876.1) was retrieved from the American Gene Database, and the homology analysis was performed by BLAST software to obtain the specific conserved target sequence of the NDM-1 gene (sequence 1 in the sequence table), and then according to The conserved target DNA sequence was designed using the software Primer Explorer V4 (http: / / primerexplorer.jp / e / ) to design primers for the detection of LAMP based on Taqman probes for the NDM-1 gene. The primer sequences are as follows:

[0105] Forward outer primer CJXJ1F3: 5'-GCATAAGTCGCAATCCCCG-3' (designed for the specific...

Embodiment 3

[0128] Embodiment 3, the LAMP detection kit based on Taqman probe

[0129] The reaction solution with the following substance concentrations: 20mM Tris·HCl (pH8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 0.1% Tween20, 0.8M betaine (betaine), 8mM MgSO 4 L, 1.4mM dNTP each, 8U BstDNA polymerase, and LAMP amplification system (double distilled water) without DNA as a negative control were packaged together to obtain a kit for LAMP detection based on Taqman probes.

[0130]

[0131]

[0132]

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Abstract

The invention provides a loop-mediated isothermal amplification (LAMP) method based on a TaqMan probe, and an LAMP primer and a kit special for the same. The loop-mediated isothermal amplification method based on the TaqMan probe, and the LAMP primer and the kit special for the same are used for detecting a target gene. According to the invention, the TaqMan probe is combined with the LAMP technology to solve the problem of nonspecific amplification fundamentally; the loop-mediated isothermal amplification method is capable of detecting the target gene quickly, conveniently and efficiently at high specificity and high sensitivity under the isothermal condition, thus providing a new technology platform for nucleic acid detection; therefore, the loop-mediated isothermal amplification method can be applied to screening and detecting pathogenic bacteria (such as superbacteria) for grass-roots medical treatment and public health units and various disease preventing and control centers, and has wide market prospect and high economic and social benefits; consequently, the loop-mediated isothermal amplification method is suitable for large-range popularization and application.

Description

technical field [0001] The invention belongs to the molecular biology detection method of nucleic acid in the field of biotechnology, in particular to a Taqman probe-based loop-mediated constant temperature amplification method and its application in the detection of target genes (nucleic acid). Background technique [0002] Real-time fluorescent quantitative PCR technology was launched by Applied Biosystems in 1996. Because this technology not only realized the leap from qualitative to quantitative PCR, but also compared with conventional PCR, it has stronger specificity and does not require post-processing of PCR products. It has the characteristics of effectively solving the problem of PCR pollution and high degree of automation, and has been widely used at present. [0003] TaqMan fluorescent probe PCR technology is a kind of real-time fluorescent quantitative PCR technology. During PCR amplification, a specific TaqMan fluorescent probe is added at the same time as a pai...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2600/166C12Q2531/119
Inventor 刘威黄留玉袁静杨展李环崔茜
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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