Method for detecting multiple target nucleic acids, a kit and application thereof

Abstract

The invention provides a method for detecting multiple target nucleic acids. The method comprises the following steps: (1) preparing a mixture required by a loop-mediated isothermal amplification reaction according to the multiple target nucleic acids, wherein the mixture comprises a primer group and a probe group which are respectively designed for multiple target nucleic acids, and each of the primer group and the probe group comprises two outer primers F3 and B3, two inner primers FIP and BIP, a loop primer, a scorpion-shaped primer probe, and nucleic acid to be detected; (2) carrying out amplification reaction on the mixture obtained in the step (1) at a constant temperature; and (3) detecting the reaction result of the step (2). The method provided by the invention has no dependence on the target nucleic acid to be detected, and the detection analysis of the target nucleic acid can be carried out by selecting a proper amplification area for each target nucleic acid and carrying out matched primer design.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a method for detecting multiple target nucleic acids. Background technique [0002] In recent years, a series of genetic detection methods have emerged in an endless stream, and the classic methods have been continuously improved. Due to the development of loop-mediated isothermal amplification (LAMP) technology, the development of genetic detection research has been greatly promoted. and apply. Loop-mediated isothermal amplification technology is a new nucleic acid-specific amplification technology established by Notomi et al. It has the advantages of strong specificity, high sensitivity, simple operation, and easy detection of products. This technique has been widely used in the field of molecular diagnostics. LAMP designs 4 core primers for 6 specific parts of the target sequence, and uses Bst DNA polymerase with strand displacement activity to catalyze the synthesis of new str...

AI Technical Summary

Problems solved by technology

The method is cumbersome to operate and requires specific kits to purify LAMP products. The sequencing process requires special personnel, as well as sequencers and sequencing reagents that cannot be afforded by ordinary laboratories.

These disadvantages limit the widespread use of this method

[0005] In addition, none of the existing multiple LAMP detection technologies can achieve rapid detection, and it takes more than 2.5 hours to complete multiple LAMP detection

Due to the high sensitivity of the LAMP reaction, the operation of uncapping the LAMP product will cause great pollution to the subsequent LAMP experiments.

[0006] In summary, LAMP in the prior art has insurmountable technical problems in the detection of single amplification or multiple amplification. Based on this, there is a need for a loop-mediated isothermal amplification method that can simultaneously detect multiple target nucleic acids.

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