Method for determining the amount of an analyte in a sample

• US 8,615,368 B2
• Filed: 03/10/2006
• Issued: 12/24/2013
• Est. Priority Date: 03/10/2005
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Light, James P. II, Carrick, James M.
• Primary Examiner(s): Lin, Jerry
• Application Number:
  US11/372,468
• Publication Number:
  US 20060276972A1
• Time in Patent Office: 2,846 Days
• Field of Search: 702/20
• US Class Current: 702/20
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2035/00346 Heating or cooling arrangements
• CPC Class Codes: G01N 2035/00356 Holding samples at elevated temperature (incubation)
• CPC Class Codes: G01N 2035/00524 Mixing by agitating sample carrier
• CPC Class Codes: G01N 2035/0097 monitoring reactions as a function of time
• CPC Class Codes: G01N 2035/0441 for samples
• CPC Class Codes: G01N 2035/0443 for reagents
• CPC Class Codes: G01N 2035/0444 for cuvettes or reaction vessels
• CPC Class Codes: G01N 2035/0455 Coaxial carousels
• CPC Class Codes: G01N 2035/0491 Position sensing, encoding; closed-loop control
• CPC Class Codes: G01N 2035/103 using disposable tips
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6454 : using an integrated detector array
• CPC Class Codes: G01N 21/76 : Chemiluminescence; Bioluminescence
• CPC Class Codes: G01N 2201/0415 : Carrusel, sequential
• CPC Class Codes: G01N 35/00693 : Calibration
• CPC Class Codes: G01N 35/0092 : Scheduling
• CPC Class Codes: G01N 35/0098 : involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: G01N 35/0099 : comprising robots or similar manipulators robots per se B25J
• CPC Class Codes: G01N 35/025 : having a carousel or turntable for reaction cells or cuvettes
• CPC Class Codes: G01N 35/026 : having blocks or racks of reaction cells or cuvettes
• CPC Class Codes: G01N 35/028 : having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/04 : Details of the conveyor system G01N35/021 - G01N35/028 take precedence
• CPC Class Codes: G01N 35/1002 : Reagent dispensers
• CPC Class Codes: Y10S 435/808 : Optical sensing apparatus
• CPC Class Codes: Y10S 435/809 : Incubators or racks or holders for culture plates or containers
• CPC Class Codes: Y10T 436/113332 : with conveyance of sample along a test line in a container or rack
• CPC Class Codes: Y10T 436/25 : including sample preparation

Abstract

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.

Claims

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.