A high-accuracy nucleic acid concentration determination method based on absorbance

Abstract

The invention discloses an absorbancy-based high-accuracy nucleic acid concentration determining method. The method comprises the following steps: (1) preparing a nucleic acid standard substance, a nucleic acid sample and a corresponding blank buffer solution; (2) using the blank buffer solution for performing gradient dilution on the nucleic acid standard substance, respectively determining the absorbancy of the standard substance at each concentration level and a to-be-tested sample after the dilution and calculating initially tested concentration c0 of the sample; (3) using the nucleic acid standard substance for preparing a pair of standard substances K1c0 and K2c0 according to the initially tested concentration, adopting a bracketing method for further determining the concentration of the nucleic acid sample, calculating respective standard uncertain degree mu1 and mu2 of the pair of standard substances according to a preparation process and further calculating a compounding standard uncertain degree uc of a determined result; (4) solving a nonlinear plan and minimizing the uncertain degree, thereby acquiring specific values of K1 and K2; (5) preparing high standard and low standard according to the calculated values of K1 and K2 and adopting the bracketing method for determining the sample, thereby acquiring an accurate result c of the sample.

Description

technical field [0001] The invention relates to the field of spectrophotometry detection, in particular to a high-accuracy nucleic acid concentration determination method based on absorbance. Background technique [0002] Metrology is an activity to achieve unit unity and accurate and reliable values. Research and establishment of high-accuracy measurement methods is one of the important contents of metrology research. Content (concentration) describes the quantity of the measured object in unit mass or volume, and is the basic quantity in chemistry and biometrics. Ultraviolet spectrophotometry is a common method for measuring nucleic acid content (concentration). It is based on the light absorption of nucleic acid in the ultraviolet region to measure nucleic acid content (concentration) according to Lambert-Beer's law. The mathematical expression of Lambert-Beer's law is as follows: [0003] A=εlc (1) [0004] in: [0005] A - Absorbance; [0006] ε——absorption coeffici...

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Problems solved by technology

[0011] Aiming at the problem that the uncertain influence is not considered in the method design and the standard curve method quantitatively introduces too large linear fitting uncertainty, the present invention establishes a high-accuracy nucleic acid quantitative method based on absorbance measurement, including the following steps :

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