Method for determining the amount of an analyte in a sample
View Patent ↗An automated analyzer for performing multiple diagnostic assays simultaneously includes multiple stations in which discrete aspects of the assay are performed on fluid samples contained in sample vessels. The analyzer includes stations for automatically preparing a sample, incubating the sample, preforming an analyte isolation procedure, ascertaining the presence of a target analyte, and analyzing the amount of a target analyte. An automated receptacle transporting system moves the sample vessels from one station to the next. A method for performing an automated diagnostic assay includes an automated process for isolating and amplifying a target analyte, and, in one embodiment, a method for real-time monitoring of the amplification process.
1. A computer-implemented method for determining the amount of an analyte of interest in a sample, the method comprising:
(a) forming amplification products from an analyte of interest;
(b) collecting signal data with a signal measuring device concurrently with the formation of amplification products, the collected signal data comprising periodic measurements of the level of a signal emitted by the sample, wherein the level of signal emitted by the sample corresponds to the amount of amplification products formed by the sample;
(c) storing the collected signal data in computer-readable media; and
(d) using a computer programmed with an algorithm, which, when executed by the computer, is configured to cause the computer to access the data stored in step (c) and to:
(1) adjust the data collected in step (b) to remove background contributions to the measured levels associated with sources other than the presence of the analyte of interest;
(2) normalize the data adjusted in step (d) (1) by dividing the adjusted data by the maximum level measured, adjusted to remove background contributions;
(3) approximate a curve through a portion of the adjusted, normalized data between a predetermined low bound stored on computer-readable media and a predetermined high bound stored on computer-readable media;
(4) determine an emergence time at which the curve approximated in step (d) (3) crosses a predetermined threshold data level, wherein the predetermined threshold data level is a level of the adjusted, normalized data that corresponds to an amount of amplification product formed in the sample; and
(5) determine the amount of the analyte of interest by comparing the emergence time determined in step (d) (4) with threshold times determined for known amounts of the analyte of interest and stored on computer-readable media, wherein the amount of the analyte of interest in the sample is unknown prior to performing steps (a)-(d).
2. The method of claim 1 , wherein the approximating step comprises applying a curve fit procedure to the adjusted, normalized data points between the predetermined low bound and the predetermined high bound.
3. The method of claim 2 , wherein the curve fit procedure is a linear least squares fit.
4. The method of claim 1 , wherein the data collected comprises measurements of the level of the signal emitted taken at least once every 30 seconds.
5. The method of claim 1 , wherein the predetermined low bound is about 0.04 and the predetermined high bound is about 0.36.
6. The method of claim 1 , wherein the normalized predetermined threshold data level is about 0.11.
7. The method of claim 1 , wherein said adjusting step comprises determining a level of background contribution and then subtracting the determined level of background contribution from the data collected in step (b).
8. The method of claim 7 , wherein determining the level of background contribution comprises a step for determining an amount of background contribution.
9. The method of claim 1 , wherein the analyte of interest is a target nucleic acid.
10. The method of claim 1 , wherein the signal emitted is a fluorometric emission.
11. The method of claim 1 , further comprising the step of isolating the analyte of interest within a sample, prior to forming amplification products.