Loop-mediated isothermal amplification (LAMP) method based on TaqMan probe, and LAMP primer and kit special for same

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 acids). 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 pa...

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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.

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