Calibration method, apparatus and computer program product

A computer program product for quantifying an analyte polynucleotide that may be present in a test sample using a nucleic acid amplification assay that comprises co-amplification, with a local instrument that amplifies nucleic acids and monitors amplicon synthesis as a function of time, of the analyte polynucleotide and a fixed amount of an internal calibrator, said computer program product comprising a tangible embodiment of software instructions for performing the steps of:

• (a) receiving a pair of coordinates for a fixed-point on a calibration curve specific for the nucleic acid amplification assay to be used for quantifying the analyte polynucleotide,
• wherein the pair of coordinates specify an amount of the analyte polynucleotide and a normalized indicia of amplification value;
• (b) obtaining a value for indicia of amplification for a known amount of the analyte polynucleotide normalized to indicia of amplification for the fixed amount of the internal calibrator contained in an adjustment calibrator and that co-amplified in a first amplification reaction performed with the local instrument;
• (c) preparing an adjusted calibration curve that comprises a first point and a second point,
• wherein the first point comprises coordinates for the known amount of the analyte polynucleotide of the adjustment calibrator and the value obtained in step (b), and
• wherein the second point comprises the pair of coordinates, received in step (a), for the fixed-point;
• (d) obtaining a value for indicia of amplification for an unknown amount of the analyte polynucleotide in the test sample normalized to indicia of amplification for the fixed amount of the internal calibrator that co-amplified therewith in a second amplification reaction performed with the local instrument;
• (e) comparing the value obtained in step (d) with the adjusted calibration curve to yield a quantitative result for the unknown amount of the analyte polynucleotide present in the test sample;
• and
• (f) outputting a tangible record of the quantitative result from step (e).

An apparatus for determining the starting quantity of a target nucleic acid sequence in a test sample, the apparatus comprising:

• (a) at least one detection mechanism that measures:
• (i) signals indicative of the respective quantities of the target nucleic acid sequence and of a first internal calibrator being amplified in a first nucleic acid amplification reaction,
• wherein the first internal calibrator comprises a second nucleic acid sequence different from the target nucleic acid sequence;
• (ii) signals indicative of the respective quantities of a known amount of an analyte polynucleotide and a second internal calibrator being amplified in a second nucleic acid amplification reaction,
• wherein the analyte polynucleotide and the second internal calibrator are both components of an adjustment calibrator,
• wherein the second internal calibrator comprises the second nucleic acid sequence, and
• wherein the starting quantity of the second nucleic acid sequence is substantially equal in the first and second nucleic acid amplification reactions;
• (b) at least one processor in communication with the detection mechanism,
• wherein the processor is programmed with a pair of coordinates for a fixed-point specifying an analyte polynucleotide amount and a normalized indicia of amplification value, and
• wherein the processor further is programmed to perform the steps of:
• (i) determining from the measured signals respective values for indicia of amplification for the known amount of the analyte polynucleotide, each of the first and second internal calibrators, and the target nucleic acid sequence;
• (ii) normalizing the indicia of amplification value determined for the target nucleic acid sequence to the indicia of amplification value determined for the first internal calibrator;
• (iii) normalizing the indicia of amplification value determined for the known amount of the analyte polynucleotide to the indicia of amplification value determined for the second internal calibrator;
• (iv) establishing a calibration curve from the fixed-point, the known amount of the analyte polynucleotide and the normalized indicia of amplification for the known amount of the analyte polynucleotide;
• and
• (v) determining the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the normalized indicia of amplification for the target nucleic acid sequence.

A method of establishing an adjusted calibration curve for a quantitative assay performed using a local instrument that amplifies nucleic acid and monitors amplicon synthesis as amplification is occurring, said method comprising the steps of:

• (a) obtaining a pair of coordinates for a fixed-point on a calibration curve specific for the quantitative assay,
• wherein the pair of coordinates specify an amount of an analyte polynucleotide and an indicia of amplification value;
• (b) obtaining an analyte polynucleotide standard that comprises a known amount of the analyte polynucleotide;
• (c) amplifying the analyte polynucleotide of the analyte polynucleotide standard in a nucleic acid amplification reaction using the local instrument;
• (d) determining indicia of amplification for the analyte polynucleotide that amplified in step (c);
• and
• (e) establishing the adjusted calibration curve by preparing a calibration plot that comprises a first point and a second point,
• wherein the first point comprises coordinates for the known amount of the analyte polynucleotide of the analyte polynucleotide standard and indicia of amplification determined in step (d), and
• wherein the second point comprises the pair of coordinates, obtained in step (a), for the fixed-point.