HRM (high resolution melt)-based multiplex LAMP (loop mediated isothermal amplification) detection method and kit

Abstract

The invention discloses an HRM (high resolution melt)-based multiplex LAMP (loop mediated isothermal amplification) detection method and kit. Standard LAMP primers and DNA fluorescence binding dye areadopted, melting is performed directly after amplification, collected melting curves are subjected to normalization or principal component analysis, and simultaneous detection of multiplex LAMP target sources can be realized. Compared with traditional melting curve analysis, the method adopts whole melting curve information instead of Tm value for identification detection; design difficulties ofmultiplex LAMP analysis primers are reduced substantially, multiple LAMP products with tiny Tm differences can be detected simultaneously, and multicolor fluorescence labeling or multiple single amplification reactions performed in space in a spaced parallel manner is avoided; the quantity of target DNA capable of being detected once simultaneously is increased substantially, identification capability is not affected by initial concentration of a template, sensitivity can reach 10 copies, and the method and the kit show excellent detection performance in complex samples and have higher practical application values.

Description

technical field [0001] The invention relates to a method for simultaneously detecting multiple DNA fragments in combination with high-resolution melting curve analysis (HRM), multivariate statistics, and loop-mediated isothermal nucleic acid amplification (LAMP), in particular to the use of high-resolution melting curves to detect multiple DNA fragments in multiple LAMPs. A method for differential identification of amplicons. Background technique [0002] Loop-mediated isothermal nucleic acid amplification (LAMP) is the most widely used and studied isothermal amplification technique due to its high sensitivity and specificity, high-speed amplification and tolerance to various inhibitors present in actual samples. However, because the LAMP amplification product does not have a fragment of a known specific size like the polymerase chain reaction (PCR) product, its gel electrophoresis has a ladder-like structure, and the band size of the amplification product cannot be observed...

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Problems solved by technology

However, traditional multiple melting curve analysis requires that the amplicons in the system have a difference of 3-5°C in melting temperature (Jiang Kan, et al. Modern Food Science and Technology, 2016.32.246; Jiang Kan, et al. Food and Machinery, 2015.31.87.), therefore, multiplex LAMP primer design is required to ensure its specificity, avoid interactions between primers, and generate amplicons with sufficient Tm differences

However, the complexity of primer design and subsequent optimization steps limit the wide application of this type of multiplex LAMP detection.

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