Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method

Abstract

The invention provides an isothermal nucleic acid amplification reaction reagent. The isothermal nucleic acid amplification reaction reagent is characterized by comprising components as follows: 100-800 mM of a Tris-HCl buffer solution, 10-150 mM of sodium chloride, 10-150 mM of potassium chloride, 10-50 mM of magnesium chloride, 5-15 mM of dithiothreitol, 5%-20% of polyvinylpyrrolidone, 10-20 mM of ATP (adenosine triphosphate), 1-5 mM of dNPTs, 10-50 mM of phosphoenolpyruvate, 500-1,500 ng / mu l of pyruvate kinase, 10-500 ng / mu l of BSA (bovine serum albumin), 25-200 pmol of each primer in a primer group, 50-200 ng / mu l of T4 bacteriophage DNA helicase gp41 protein, 100-500 ng / mu l of streptomyces coelicolor recA protein, 200-1,000 ng / mu l of single-strand binding protein and 50-200 ng / mu l of escherichia coli DNA polymerase I. The invention further provides an isothermal nucleic acid amplification method. According to the isothermal nucleic acid amplification reaction reagent and the isothermal nucleic acid amplification method, nucleic acid amplification under the isothermal condition at the lower temperature is realized, and a traditional nucleic acid amplification reaction process is greatly simplified.

Description

technical field [0001] The invention belongs to the field of molecular biology. Specifically, the invention relates to a high-sensitivity isothermal nucleic acid amplification reaction reagent and an isothermal nucleic acid amplification method capable of realizing rapid amplification under isothermal conditions. Background technique [0002] Polymerase chain reaction (PCR) technology has been widely used in life science research since it was invented by Mullis et al. in 1983. In the traditional PCR reaction, in the presence of a reaction mixture of DNA templates, primers, four dTTPs, and an appropriate buffer, the target DNA fragment is amplified by DNA polymerase catalysis. The PCR reaction generally includes three steps of denaturation, annealing and extension, and each step is repeated 30 to 40 times, so that the exponential amplification of a small amount of target DNA fragments has reached a detectable level. Because PCR technology can amplify a small amount of nuclei...

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Problems solved by technology

[0004] However, due to temperature changes and specific temperature conditions during the thermal cycle, the traditional PCR technology requires the instrument to have a sophisticated temperature control program, which can accurately raise and lower the temperature at a predetermined time, so as to realize the cyclic change of temperature, which greatly increases the instrument temperature. The development cost makes the instrument expensive, and since the temperature change and maintenance are completely dependent on the power supply, the power consumption is very large

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