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Systems and methods for calibration using dye signal amplification

This application claims priority to U.S. Provisional Patent Application No. 60/898,064, filed Jan. 29, 2007, entitled “Systems and Methods for Optical and Spectral Calibration of Real-Time PCR Instrumentation,” which is incorporated herein in its entirety by reference.

The present application relates to biological testing devices, systems that contain such devices, and methods that use such devices and/or systems.

Real-time polymerase chain reaction (RT-PCR) technology, as presently practiced, relies upon the accurate detection of fluorescent emission signals above an initial baseline. The baseline signal can represent a combination of spurious or unwanted signal contributions such as the residual fluorescence contributed by the plastic or other material of a sample plate, the fluorescence of a buffer or other non-reactant liquid material, noise in the optical detector or detection electronics, or other sources of background signal noise or detection floor that are not a product of the desired PCR amplification. In various known RT-PCR implementations, better accuracy in the detection of an amplification signal, and hence original sample quantity, is frequently sought by characterizing the baseline floor over the first few PCR cycles, and then subtracting the baseline from the detected emissions once an inflection point into the exponential region has been reached.

Known calibration techniques for calibrating or adjusting the detected emission data captured by a RT-PCR or other instrument can involve calibration based on a plate loaded with a pure dye formulation but no sample material. A RT-PCR detector can, for instance, take emission readings from a standard 96-well sample plate or other support, to determine intensity range or spectral behavior, and perform a calibration or normalization based on those values. Such calibration techniques, however, depend on the emission data from a reference dye or other material under static conditions. Moreover, the calibration factors generated by known pure-dye calibration techniques can be limited to fixed scaling factors or other adjustments, whereas the intensity range and spectral behavior of actual emission data can possibly vary with time under the varying cycles and conditions of an RT-PCR or other analytic process. Reference calibration does not match actual performance calibration due to real time running effects, such as thermal cycling, condensation, evaporation, chemistry changes, and the like. A need exists for calibration and related techniques that address these and other issues.

Systems and methods according to various embodiments of the present teachings, relate to techniques and platforms to capture, identify, and calibrate emission data of an RT-PCR or other amplification reaction, on a real-time basis in which the reference dye, actual RT-PCR reactions, or other material can itself be subjected to RT-PCR thermal cycling or other dynamic processing. According to various embodiments, the reference dye can be subjected to the same number and type of RT-PCR thermal cycles as a sample being tested is exposed to, and can produce a time-varying emission output. The emission output data can be captured and stored across the entire set of cycles of the RT-PCR or other process. The detected intensity range, spectral response, and other characteristics of the amplified dye or other material can be used to generate a calibration curve, function, or other calibration data set whose ranges, limits, or other parameters can vary with time and be set to correlate with different cycles in an RT-PCR, or other assay or reaction. As a result, better calibration accuracy over the entire RT-PCR process can be achieved. In some embodiments, the calibration occurs during the actual amplification reaction that is sought to be calibrated such that even minor differences due to DNA sequence construction, pH, and other variables that affect the spectrum of a dye can be accounted for.

FIG. 1 illustrates an exemplary PCR amplification profile or curve, according to various embodiments of the present teachings.

FIG. 2 illustrates a schematic of a PCR detection system, according to various embodiments of the present teachings.

FIG. 3 illustrates a calibration data set for a PCR amplification profile or curve, according to various embodiments of the present teachings.

FIG. 4 illustrates a flowchart of calibration processing, according to various embodiments of the present teachings.