Fluorescent isothermal nucleic acid amplification method

Abstract

The invention discloses a fluorescent isothermal nucleic acid amplification method. The amplification method comprises the following ingredients: a Tris buffer, potassium acetate, magnesium acetate, dithiothreitol, polyethylene glycol, ATP, dNTPs, creatine phosphate, a single-chain binding protein, a recombinant enzyme, a UvsY protein, a DNA polymerase, an exonuclease, a fluorescent probe and a primer group. The method can be applied to carry out real-time fluorescence monitoring on DNA; and based on the method, addition of an RNA retrovirus cane realize RNA amplification. The isothermal nucleic acid amplification method comprises the steps as follows: (1) extracting DNA or RNA; (2) adding the DNA or RNA to a nucleic acid amplification system; and (3) mixing evenly, adding the mixture into a fluorescence detection instrument, reacting at 35-45 DEG C for 5 to 30 minutes to observe a fluorescence signal. The method has the advanategs of simple operation, fast response, low requirements for operators, and broader range of applications.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to a fluorescent isothermal nucleic acid amplification method, which realizes the fluorescent isothermal nucleic acid amplification at 35-45°C. Background technique [0002] Since 1983, the polymerase chain reaction (PolyChainReaction, PCR) technology invented by KaryMullis has made a breakthrough revolution in the field of molecular biology. Nucleic acid amplification detection technology represented by PCR has developed rapidly. After decades of development, PCR technology has been widely used in various fields of life sciences such as molecular biology, medicine, law, etc. due to its outstanding advantages such as sensitivity, specificity, speed, simplicity, and ease of automation. [0003] The process of PCR consists of three basic reaction steps of denaturation-annealing-extension. Generally, the denaturation temperature is 93-96°C, the annealing temperat...

AI Technical Summary

Problems solved by technology

Since PCR requires constant temperature changes to achieve nucleic acid amplification, it has been unable to get rid of the limitation of relying on sophisticated instruments and equipment since its birth; moreover, during the operation of PCR, it needs professionals to operate, and the requirements for personnel are relatively high; in addition , the PCR amplification time is generally completed within a few hours, which does not meet the needs of rapid detection. In view of this, the PCR-based nucleic acid amplification detection technology cannot be widely applied and promoted outside the laboratory.

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