MTAS^® Optical

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qPCR thermocyclers

qPCR thermocyclers have a heat cycling block or reaction chamber like a regular thermocycler, with the addition of an optical detection unit. This optical detection unit consists of a light source, optical filters, lenses, as well as an optical detector. Therefore qPCR thermocyclers can detect small amounts of fluorescence and/or light from either a wide spectrum and/or a defined smaller spectrum. The detection spectrum may vary between 400 and 720 nm.

The reason for Optical Calibration of qPCR thermocyclers

Chemistry reaction factors
Temperature performance of reaction block
Temperature performance of the heated lid
Lenses, mirrors and optical pathway(s)
Sensitivity of optical detector
Mechanical tolerances of mechanisms in place
Signal processing parameters of the software, as base-line, base line correction, S/N ratio detection etc.
To have an understanding if and how results are influenced by “non primary chemistry factors” qPCR thermocyclers should be calibrated for temperature and optics.

High Resolution Melt Modules (HRM)

A High Resolution Melt module partly consists of a non-cycling heat block or reaction chamber which slowly increases temperature while in operation. The module also contains an optical detection unit. This optical detection unit consists of a light source, optical filters, lenses, as well as an optical detector. HRM modules can detect small amounts of fluorescence and/or light from either a wide spectrum and/or a defined smaller spectrum. The detection spectrum may vary between 400 and 720 nm.
Detected fluorescence changes, occurring during the melt track of the amplicon is processed by software of the qPCR thermocycler.
In general qPCR thermocyclers generate and compare relative values rather than absolute values.
Detected values are compared to assigned values of designated wells. The generated values are an outcome of measured and processed data where the processing parameters are typically insufficiently described or documented.

Currently used Optical Calibration Methods

Biological test plates with one or multiple dyes (non traceable)

Dye (crystal) test plates (non traceable)

Melt test plates (non traceable)

Combined Temperature-Optical Probe Plate (traceable)

Items measured with patented Combined Temperature-Optical Probe Plate

Calibrated Traceable Temperature Items for (q)PCR Thermocyclers

Multi Channel, 4Hz, static temperatures, dynamic temperatures, accuracy, uniformity, static and dynamic, ramp rates, overshoots, undershoots, hold times, heated lid temperature and environmental conditions.

Calibrated Traceable Optical Items for (q)PCR Thermocyclers

Multi Channel, Cq detection (uniformity and accuracy), optical saturation, detection linearity, melt curve accuracy, melt curve uniformity, ratio Tm and linear sensitivity factor.

The qPCR thermocycler program used when calibrating with the Combined Temperature-Optical Probe Plate

Cycler program

qPCR Thermocycler program (Graphical)

So actually the CU is emulating, depending on the measured temperature, a typical Absolute Quantification qPCR light intensity profile. A schematic view is showed below.

The graph can be divided into flowing sequential partitions:

Base Line partition
S/N partition (Signal / Noise)
Linear partition
Exponential partition
Saturation partition

What are the Traceability units Measured or generated by Calibration Unit (CU)

Temperature in: °C (ITS-90)
Light and intensity in W/sr: I_e , Radiant Intensity [W/sr] watt per steradian (squared radian) (Radiometric Flux per unit solid angle)
Description: Radiant power of a source emitted in a certain direction

Quantification cycle (Cq), Cq detection

The qPCR Thermocycler analyses the by the CU generated, "light intensity" and calculates Cq values for each channel. The CU provides identical signals for the first 10 cycles, as part of the "base line partition". After 10 cycles the CU start slowly increasing "light intensity" whereas the DUT has to pickup any "light intensities" passing the DUT's minimum detection level. The DUT can than detect and calculate a Cq value for that channel. An ideal cycler with ideal optics should therefore present analyses with identical Cq values for each channel. Cq values are expressed as the number of qPCR cycles required to reach a set minimum "light" signal level.

Validation of Cq values

Cq values should be identical for each channel. The laboratory must define how much tolerance between Cq values are acceptable. For example the laboratory may accept a maximum tolerance of 10 %. Additionally, verification can be made by comparing the detected absolute Cq values with Cq values of the same type of equipment (brand and model) which gives an indication how the tested qPCR Thermocycler compares with similar equipment. (CYCLERtest "market specs")

Saturation Partition

Raw data collected by the DUT at the final saturation signal (100 %) provided by the CU is used to determine the linear signal sensitivity of the DUT. (see also Linear Sensitivity Factor)

Melting Point

While the qPCR thermocycler (DUT) is slowly increasing its block temperature, the optical unit of the DUT monitors changes in light intensity. The Calibration Unit (CU) which is positioned in the DUT dynamically measures the temperature of each individual channel. The measured temperature is processed and proportionally converted into a defined amount of light. Defined thresholds of temperature readings are set as a threshold for the release of the designate amounts of light intensity.
For an ideal qPCR thermocycler, each well is identical in temperature and identical optical readings are detected at each channel. Ideally there would be one peak, where all the signals of all channels overlay each other and the signal intensity detected by the qPCR thermocycler should be identical for all channels. Melting points are expressed as Tm and in ºC.

Melting Point Bias

As mentioned previously Melting points are expressed as Tm and in ºC. The melting point calculated by the DUT can differ from the set melting point standard from the CU. The Calibration Unit (CU) which is positioned in the DUT dynamically measures the temperature of each individual channel and converts each temperature into a defined intensity of light for each individual channel. The default set start temperature for emulating the individual channel Tm is 70.0 ºC whereas that total melt track is 0.2 ºC. For an ideal qPCR thermocycler, each well is identical in temperature and identical optical readings are detected at each Ideally one should see one peak, where all the signals of all channels overlay each other and a Tm calculated by the DUT would be 70.1 ºC. The deviation of the set Tm (CU) and the found Tm (DUT) is called bias of Tm (bias Tm). If the bias Tm of two or more different DUT’s are known one is able to compare and normalize generated results of DUT’s by adjusting for the known bias.

To normalize results:

Peak Shifts

Since the calibration unit (CU) measures temperature for each channel and converts at defined temperature thresholds to light intensities, melt curve peak shifts will occur. In the case of ideal optics, light detection and analyzing software of the DUT, peak shifts will be identical to the temperature uniformity of the qPCR thermocycler as measured by the CU. In the case of an ideal qPCR thermocycler with perfect temperature uniformity, ideal optics, light detection, and analyzing software, there would be only one peak where all channels overlay one another.

Peak Heights

The calibration unit (CU) converts temperature to traceable and pre-defined intensities of light. Melt curve peaks are detected by the thermocycler optics. In case of ideal optics, light detection and analyzing software of the DUT, peak heights (measured intensity of light) will be identical for each CU channel. Differences in peak heights indicate differences in optical pathway, optics, optical detection and sensitivity of the DUT.

Channel Peak Height Consistency (CPHC)

Peak heights should be within pre-defined thresholds. Individual peak heights are expressed as Channel Peak Height Consistency (CPHC)

Calculation of CPHC (Channel Peak Height Consistency)

For an ideal qPCR thermocycler, where each well is identical in temperature and where identical optical readings are detected at each well, there should be one peak, where all the signals of all channels overlay each other and the signal amount detected by the qPCR thermocycler should be identical for all channels.
In the case of an ideal qPCR thermocycler the CPHC will be 1.00

Interpretation:

Linear Sensitivity Factor (LSF)

During the Optical Calibration, the CU supplies defined intensity amounts of light with a known spectrum to the DUT (qPCR thermocycler) The CU provides a maximum intensity of 100 % (saturation phase) whereas the decrease to 60 % of intensity from saturation to "prior melt" is equal (?40 %) to the instant quench of intensity occurring during melt and after the melt to 20 % intensity (?40 %) offered by the CU. Calculating the ratio between a range of provided signal intensities of the CU and the detected intensities of the DUT enables the calculation of a linear sensitivity factor for each channel.

Validation of Tm (RTm or Ratio Tm)

Validation of Tm can be based on the absolute values, analyzed by the DUT, as well as calculating the ratio between the delta Tm analyzed (?Tm DUT) of all channels and the temperature uniformity during the Tm-set of the CU. (?t Tm CU)

Tm is measured and calculated by the DUT and t(max) and t(min) @ Tm is measured by the CU.
The RTm value should be within predefined specs and/or market specs.

A qPCR thermocycler with ideal optics and analyzing software would end up with a RTm value of 1.0, regardless of any deviation in absolute block temperature and block uniformity.

RTm =1 and within specs

RTm > 1 and outside maximum specs

Tm Peak Shift Fact (TPSF) and Identical Fragment Indicator (IFI)

The Identical Fragment Indicator (IFI) is a precise calculation to determine if the DUT detected Tm range is solely temperature related or not.

S/N ratio detector (SNR)

SNR detector calculates the average noise of the base line to determine the minimum detection limit of the DUT. The SNR calculated from the raw data collected by the DUT is expressed as the average of the base line plus 3 times it's calculated standard deviation. (Signal to Noise Ratio is = 3) The SNR is expressed as a cycle number. The SNR value can be compared to the Cq value analyzed by the DUT. SNR and Cq values should be close to each other whereas the absolute difference between SNR and Cq value should be identical for each channel. Differences found in Cq and SNR values are indicators for the sensitivity of the DUT and its software settings. Channel differences of SNR and Cq values indicated incorrect base-line settings and/or calculation of the DUT software.

Measured Parameters when the DUT is lacking Dissociation ability

If the DUT is lacking the ability to execute a dissociation curve (melting curve) a number of parameters cannot be determined. Consequently the Calibration Certificate will contain less information as some parameters are calculated using different data points.

Optical Calibration in High Advance Mode

In addition to the already described calibration parameters the calibration unit (CU) has an Advanced Mode for further investigations of the qPCR thermocycler.

Optical bandwidth calibration DUT
Optical channel “crosstalk” calibration DUT
Excitation Spectrum and Intensity Calibration DUT
S/N ratio detector (SNR)

The Spectrum provided by the CU to the DUT

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