Calibration Method, Apparatus and Computer Program Product

Abstract

Method and system for quantifying target nucleic acids using real-time amplification and internal calibration adjustment. The approach employs a single fixed data point in combination with a single adjustment calibrator amplified on the instrument that is to be calibrated for approximating a complete calibration curve.

1 .- 31 . (canceled) 
     
     
         32 . A calibrated system for determining the starting quantity of a target nucleic acid sequence in a test sample, the calibrated system comprising:
 (a) a local instrument that carries out real-time nucleic acid amplification, the local instrument comprising a temperature-controlled incubator and a detection system,
 wherein the detection system is configured to measure signals indicating respective quantities of a known amount of an analyte polynucleotide and an internal calibrator polynucleotide that coamplify in a real-time nucleic acid amplification reaction carried out using the temperature-controlled incubator, 
 wherein the analyte polynucleotide and the internal calibrator polynucleotide are both components of an adjustment calibrator; and 
   (b) a processor in communication with the detection system of the local instrument,
 wherein the processor is programmed with a pair of coordinates for a fixed-point specifying an amount of the analyte polynucleotide and a first normalized indicia of amplification value, wherein the fixed-point was determined from a linear calibration curve fitted to pooled data that was not determined using the local instrument, 
 wherein the processor is programmed with software causing the processor to determine indicia of amplification for each of the analyte polynucleotide and the internal calibrator polynucleotide that coamplify in the real-time nucleic acid amplification reaction, and then divide one by the other to calculate a second normalized indicia of amplification value, 
 wherein the processor is further programmed with software causing the processor to fit a linear calibration curve to the fixed-point and a pair of coordinates specifying the known amount of the analyte polynucleotide of the adjustment calibrator and the second normalized indicia of amplification value, and 
 wherein the processor is further programmed with software causing the processor to compare a third normalized indicia of amplification value with the linear calibration curve to determine the starting quantity of the target nucleic acid sequence in the test sample. 
   
     
     
         33 . The calibrated system of  claim 32 , wherein the temperature-controlled incubator is configured to maintain a substantially constant temperature, and wherein the real-time nucleic acid amplification reaction is a real-time isothermal nucleic acid amplification reaction. 
     
     
         34 . The calibrated system of  claim 33 , wherein signals measured by the detection system are measured as a function of time. 
     
     
         35 . The calibrated system of  claim 34 , wherein the analyte polynucleotide and the internal calibrator polynucleotide of the adjustment calibrator are synthetic transcripts. 
     
     
         36 . The calibrated system of  claim 34 , wherein the detection system detects optical signals from one or more fluorescent labels. 
     
     
         37 . The calibrated system of  claim 36 , wherein the detection system comprises a fluorometer. 
     
     
         38 . The calibrated system of  claim 37 , wherein the fluorometer comprises a visible light excitation light source. 
     
     
         39 . The calibrated system of  claim 36 , wherein optical signals received by the detection system are converted into signals that can be operated on by the processor. 
     
     
         40 . The calibrated system of  claim 36 , wherein the processor is programmed with software causing the processor to determine threshold-based indicia of amplification. 
     
     
         41 . The calibrated system of  claim 40 , wherein the processor is a freestanding computer that is not physically joined to the local instrument. 
     
     
         42 . The calibrated system of  claim 40 , wherein the processor is in communication with the detection system via either a wired connection or a wireless connection. 
     
     
         43 . The calibrated system of  claim 42 , wherein the local instrument and the processor are both parts of an integrated assembly on a single chassis. 
     
     
         44 . The calibrated system of  claim 43 , wherein the processor comprises a memory component that stores a tangible record of the linear calibration curve. 
     
     
         45 . The calibrated system of  claim 43 , further comprising at least one of a user interface, a keyboard, and an output device for data display. 
     
     
         46 . The calibrated system of  claim 32 , wherein the real-time nucleic acid amplification reaction is carried out using thermal cycling to amplify nucleic acids, and wherein signals measured by the detection system are measured as a function of reaction cycle number. 
     
     
         47 . The calibrated system of  claim 46 , wherein the temperature-controlled incubator comprises a temperature-controlled block configured to hold either a plurality of reaction vessels or a multiwell plate. 
     
     
         48 . The calibrated system of  claim 32 , further comprising at least one of a user interface, a keyboard, and an output device for data display. 
     
     
         49 . The calibrated system of  claim 32 , wherein the processor comprises a memory component that stores a tangible record of the linear calibration curve. 
     
     
         50 . The calibrated system of  claim 32 , wherein the real-time nucleic acid amplification reaction is carried out isothermally, and wherein signals measured by the detection system are measured as a function of time. 
     
     
         51 . The calibrated system of  claim 32 , wherein the temperature-controlled incubator comprises a temperature-controlled block configured to hold either a plurality of reaction vessels or a multiwell plate. 
     
     
         52 . The calibrated system of  claim 32 , wherein the detection system detects optical signals from one or more fluorescent labels. 
     
     
         53 . The calibrated system of  claim 52 , wherein the detection system comprises a fluorometer. 
     
     
         54 . The calibrated system of  claim 53 , wherein the fluorometer comprises a visible light excitation light source. 
     
     
         55 . The calibrated system of  claim 52 , wherein optical signals received by the detection system are converted into signals that can be operated on by the processor. 
     
     
         56 . The calibrated system of  claim 32 , wherein the processor is a freestanding computer that is not physically joined to the local instrument. 
     
     
         57 . The calibrated system of  claim 32 , wherein the processor is in communication with the detection system via either a wired connection or a wireless connection. 
     
     
         58 . The calibrated system of  claim 32 , wherein the local instrument and the processor are both parts of an integrated assembly on a single chassis. 
     
     
         59 . The calibrated system of  claim 32 , wherein the processor is programmed with software causing the processor to determine threshold-based indicia of amplification. 
     
     
         60 . The calibrated system of  claim 32 , wherein the analyte polynucleotide and the internal calibrator polynucleotide of the adjustment calibrator are synthetic transcripts.