Novel method for isothermal amplification detection of double-stranded nucleic acid based on nicking enzyme

Abstract

The invention relates to a novel method for isothermal amplification detection of a double-stranded nucleic acid based on nicking enzyme, belongs to the field of nucleic acid test, and in particular to a method for amplifying a single-chain target from a double-stranded nucleic acid target under the combined action of the nicking incision enzyme and polymerase and carrying out efficient specificity amplification on the target nucleic acid employing two amplification products and a replacement primer in an isothermal condition. Through selection of one amplification primer and an amplification template annealing region, efficient specificity amplification of the target nucleic acid can be finished by only three primers; the 3' end of the template is completely complementary with the primer; the specificity is improved; meanwhile, the problem of refractory amplification due to the fact that the replacement primer occupies the complementary position of the amplification primer in the prior art is avoided; the amplification primer can also be designed into a form of a molecular beacon; and only correctly amplified target sequence can be annealed together with the primer in the form, and generates subsequent fluorescence signals, so that the method has relatively good specificity.

Description

technical field [0001] The invention relates to nucleic acid isothermal amplification and detection technology, and belongs to the field of nucleic acid detection. More specifically, it relates to a method for efficiently and specifically amplifying a target nucleic acid under isothermal conditions by using two amplification primers and one displacement primer under the joint action of a nicking endonuclease and a polymerase. [0002] The invention also relates to the application in the detection of pathogenic microorganism nucleic acid. Background technique [0003] In recent decades, with the invention and application of the polymerase chain reaction (Polymerase Chain Reaction, PCR), the development of double-stranded nucleic acid in vitro amplification and in vitro detection has been greatly promoted. Because of its high sensitivity and good specificity, it has been widely used in the field of nucleic acid amplification and molecular detection, and is currently the most ...

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Problems solved by technology

[0007] In order to improve the shortcomings of the technologies described in patents US2008096257A1 and US2009092967A1, such as complex reaction systems that easily cause non-specific amplification, replacement primers occupying templates to block amplification and reduce amplification efficiency, the present invention provides a simple and efficient nucleic acid isothermal Amplification detection method, this method can realize high-efficiency and specific amplification of target nucleic acid with only three primers, and the system is simple; the design of the amplification primer P2 and the single-stranded template annealing region next to the cutting point makes the reaction have better specificity, and The 3' end of the template is completely complementary to the primer, so that the amplified template can be efficiently used by the primer, and the reaction efficiency is higher; different nicking endonucleases can be selected according to the reaction temperature, and the applicable temperature range is wide, which is suitable for on-site rapid detection needs

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