Nucleic acid amplification signal acquisition and signal analysis

A computer-implemented method for determining a value representative of an amount of a nucleic acid sequence in a sample, comprising:

• performing, by a real-time amplification detection system, continuous, non-cyclic isothermal amplification of a sample comprising a nucleic acid sequence, a sub-sequence of the nucleic acid sequence, or a complementary sequence of the nucleic acid sequence;
• and a fluorescent dye;
• repeatedly measuring, by the real-time amplification detection system, a signal comprising light emitted from the fluorescent dye;
• recording, by the real-time amplification detection system, isothermal amplification data comprising a plurality of data points, each data point comprising (i) a measured signal;
• and (ii) a time that the signal was measured, wherein said recording is performed at a frequency sufficient for statistically significant biochemical reaction rate determination;
• receiving, by a computer having at least one central processing unit including at least one microprocessor configured to execute instructions stored on at least one non-transitory computer-readable medium, the isothermal amplification data recorded by the by the real-time amplification detection system;
• performing first processing, by the computer, of the isothermal amplification data to determine whether amplification has occurred, wherein the first processing comprises:
• dividing, by the computer, the isothermal amplification data into multiple sub-segments of predetermined size;
• dividing, by the computer, each sub-segment equally into proximal signal data comprising earlier data points and distal signal data comprising later data points;
• determining, by the computer, a segment ratio for each sub-segment by determining a sum of the distal signal data and a sum of the proximal signal data, and dividing the sum of the distal signal data by the sum of the proximal signal data;
• determining, by the computer, a maximum segment ratio, and dividing the isothermal amplification data into a baseline segment and a plateau segment separated at the position of the maximum segment ratio;
• dividing, by the computer, the baseline segment and the plateau segment into proximal and distal halves, and determining a first mode comprising the statistical mode of the proximal half of the baseline segment and a second mode comprising the statistical mode of the distal half of the plateau segment;
• determining reaction amplitude, comprising the difference between the first mode and the second mode;
• when the amplitude is determined not to be greater than a predetermined threshold, determining, by the computer, that no amplification has occurred;
• and
• when the amplitude is determined to be greater than the predetermined threshold, determining, by the computer, that amplification has occurred;
• when amplification is determined not to have occurred, ending the method;
• when amplification is determined to have occurred, performing second processing, by the computer, of a logarithmic transformation of the isothermal amplification data, identifying an initial point;
• performing third processing, by the computer, of the logarithmic transformation of the isothermal amplification data, selecting a current region around the initial point, and fitting a linear model to the current region;
• performing fourth processing, by the computer, of the logarithmic transformation of the isothermal amplification data, determining a first correlation coefficient for the current region;
• extending the current region by a defined step size;
• determining a second correlation coefficient for the extended region;
• and comparing the first correlation coefficient and the second correlation coefficient;
• when a difference between the first correlation coefficient and the second correlation coefficient does not exceed a set maximum, defining, by the computer, the extended region as the current region and repeating said extending;
• when the difference between the first correlation coefficient and the second correlation coefficient exceeds the set maximum, defining, by the computer, the extended region as a selected data subset;
• determining, by the computer, values of predefined statistical measures for the data subset, including initial and final time points delimiting the data subset, number of data points contained in the data subset, and values for predefined parameters of the model;
• storing, by the computer, the data subset and the statistical measures in a database comprising at least one non-transitory computer-readable medium;
• determining, by the computer, the value representative of the amount of the nucleic acid sequence in the sample based on the values for the parameters of the model;
• and
• displaying to a user, by the computer, the value representative of the amount of the nucleic acid sequence in the sample.