Fluorescent ring-mediated isothermal amplification method based on molecular beacon

Abstract

The invention discloses a fluorescent ring-mediated isothermal amplification method based on a molecular beacon. The amplification method introduces a molecular beacon into the LAMP technology, wherein the fluorescent ring-mediated isothermal amplification method based on the molecular beacon is established and named as MB-LAMP, thereby realizing direct detection of amplified products, and fundamentally solving the problem of non-specific amplification of the LAMP method, great application value and commercial value are achieved.

Description

technical field [0001] The invention relates to a fluorescent ring-mediated isothermal amplification method based on a molecular beacon. Background technique [0002] Loop-mediated isothermal amplification technology is a new in vitro nucleic acid amplification detection technology established by Notomi in 2001. It uses 4-6 primers to identify 6-8 specific nucleic acid fragments. DNA polymerase has the function of strand displacement. With its help, the target gene can be amplified efficiently and specifically in a short period of time under constant temperature conditions. At the same time, a large amount of white magnesium pyrophosphate precipitates from the reaction. Using this feature, the experimenter can judge whether the result is negative or positive by observing the turbidity of the reaction system. Since the LAMP reaction is carried out under constant temperature conditions, a stable heat source (such as a water bath or a metal bath) can meet the requirements of ...

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Problems solved by technology

However, in addition to the amplification products, SYBR Green I can bind to all double-stranded DNA and emit light, resulting in difficult interpretation of the results and high fluorescence background values

In addition, since SYBRGreen I needs to open the reaction tube before adding after the reaction, the reaction cannot be carried out in a closed system, resulting in aerosol pollution.

[0006] 3. Precipitation method: Magnesium pyrophosphate precipitates will be produced during the LAMP reaction process, and the amount produced is relatively large. After the reaction is completed, all the precipitates can be gathered together by centrifugation. The result can be judged by visual observation, but due to coke Magnesium phosphate has very good dispersibility in the solution system, and it can be redispersed into the reaction solution in an instant after centrifugal aggregation, which is easy to produce false negatives

[0007] 4. Hydroxynaphthol blue staining (HNB): Hydroxynaphthol blue staining is a metal ion indicator, which can 2+ Different colors show different colors, due to the precipitation of magnesium pyrophosphate in the LAMP reaction, a large amount of Mg will be consumed 2+ , so that the color of HNB will change, but HNB is not stable, it is not easy to save, it needs to be used now, and it is inconvenient to use. Another problem is that the color reaction of HNB is not obvious, and it is not easy to detect the color change. Although many researches people are using this method, but it has not been recognized by everyone

[0011] None of these indirect detection methods can directly determine the amplification product. If non-specific amplification occurs, it will not be recognized. This is also a big problem encountered by LAMP technology.

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