REAL-TIME THERMOCYCLER WITH ADJUSTABLE EXCITATION UNIT

Abstract

The present disclosure provides a real-time thermocycler, comprising: a well for storing a sample comprising a target and fluorescence molecules, a thermal unit for adjusting a temperature of the sample, an excitation unit for exciting the fluorescence molecules of the sample via radiation, a detection unit for detecting a fluorescence signal from the sample, and a controller for controlling the excitation unit to adjust an intensity of the excitation of the sample based on information about the target, such that the fluorescence signal is in a working range of the detection unit.

Claims

1. A real-time thermocycler, comprising: a well for storing a sample comprising a target and fluorescence molecules, a thermal unit for adjusting a temperature of the sample, an excitation unit for exciting the fluorescence molecules of the sample via radiation, a detection unit for detecting a fluorescence signal from the sample, and a controller for controlling the excitation unit to adjust an intensity of the excitation of the sample based on information about the target, such that the fluorescence signal is in a working range of the detection unit.

2. The real-time thermocycler of claim 1, wherein the controller is configured to adjust the excitation based on information about a type of the target obtained from a database.

3. The real-time thermocycler of claim 2, further comprising a graphical user interface for entering information about the type of the target in the well in the database.

4. The real-time thermocycler of claim 1, wherein the excitation unit comprises at least two emitters, in particular at least two light-emitting diodes.

5. The real-time thermocycler of claim 1, wherein the controller is configured to determine an adjustment of the intensity of the excitation of a current sample based on a previously detected fluorescence signal from a previous sample comprising a same type of target as the current sample.

6. The real-time thermocycler of claim 1, wherein the excitation unit and/or the detection unit are mounted on a moveable arm that is configured to move the excitation unit and/or the detection unit between a plurality of wells.

7. A method for thermally cycling and detecting a first sample comprising a first target and fluorescence molecules and a second sample comprising a second target and fluorescence molecules, the method comprising: thermally cycling the first sample and exciting the fluorescence molecules of the first sample with radiation of a first excitation intensity, and thermally cycling the second sample and exciting the fluorescence molecules of the second sample with radiation of a second excitation intensity, wherein the second excitation intensity is chosen differently from the first excitation intensity based on information about the first and the second target, such that a fluorescence signal of the second sample is in a working range of a detection unit.

8. The method of claim 7, further comprising: obtaining information about the first and the second target, storing the obtained information about the first and the second target in a database, and adjusting the excitation intensity based on the information from the database.

9. The method of claim 8, wherein the obtaining the information about the first and second sample is performed by performing a measurement of the first and the second target, wherein in particular the performing the measurement includes measuring a fluorescence signal from the first and/or second sample.

10. A non-transitory computer-readable storage medium having instructions stored thereon that, when executed by at least one computing device cause the at least one computing device to perform operations, the operations comprising: thermally cycling and detecting a first sample comprising a first target and fluorescence molecules and a second sample comprising a second target and fluorescence molecules; thermally cycling the first sample and exciting the fluorescence molecules of the first sample with radiation of a first excitation intensity, and thermally cycling the second sample and exciting the fluorescence molecules of the second sample with radiation of a second excitation intensity, wherein the second excitation intensity is chosen differently from the first excitation intensity based on information about the first and the second target, such that a fluorescence signal of the second sample is in a working range of a detection unit.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] To illustrate the technical features of embodiments of the present invention more clearly, the accompanying drawings provided for describing the embodiments are introduced briefly in the following. The accompanying drawings in the following description are merely some embodiments of the present invention, modifications on these embodiments are possible without departing from the scope of the present invention as defined in the claims.

[0053] FIG. 1 is a block diagram illustrating a real-time thermocycler in accordance with an embodiment of the present invention,

[0054] FIG. 2 is a block diagram illustrating a further real-time thermocycler in accordance with a further embodiment of the present invention, and

[0055] FIGS. 3a and 3b are diagrams of experimental results obtained with real-time thermocyclers in accordance with the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

[0056] The foregoing descriptions are only implementation manners of the present invention, the scope of the present invention is not limited to this. Any variations or replacements can be easily made through person skilled in the art. Therefore, the protection scope of the present invention should be subject to the protection scope of the attached claims.

[0057] For real-time thermocyclers, the relevant information and the result are typically not contained in one measurement. There are several periodic measurements, typically between 30 and 50, to monitor a change in signal during amplification. The change of the signal over time shows the required result. Typically, the single measurements are plotted in a graph showing the signal over the cycle or time. The single measurement points are linked and sometimes fitted to form a curve.

[0058] In general, a positive result of a PCR shows a more or less sigmoidal form. There is a baseline fluorescence at the beginning, a rise of the signal and the end fluorescence. The signal rises as soon as the number of amplified DNA is so high that a signal can be detected. This is important for the analysis of data. A rise shows the presence of a target DNA and furthermore from the moment of the rise conclusions can be drawn to the amount of sample. If this rise is early, there was a high amount of target DNA at the beginning. A late rise of signal shows a small starting amount. It takes longer until there is amplified enough target DNA. After the rising point, the amplification of DNA continues and the signal rises further. The rise becomes a steady signal, when the amplification is almost finished and the reaction chemistry is used up. If there is no target DNA in the sample, there will be no rising of the signal and therefore a steady horizontal line as result.

[0059] For analysis the offset (baseline) of the resulting curve may be subtracted and further calculations can be done to smooth the curves. The subtraction of the offset can be done for easier comparison of the curves. If they all start at zero, they can be analyzed e.g. via setting a threshold.

[0060] The combination of an optical unit with a thermal unit realizes a real-time cycler for amplification and detection of DNA molecules. Amplification of the specific nucleic acid is performed in small reaction vessels called wells. Typically, eight times twelve wells are arranged in a pattern to form a 96-well microtiter plate adapter. There are instruments with other numbers or configurations of wells and there are instruments which use individual tubes. Two important units of a real-time thermocycler are the optical and the thermal unit. Of course a real-time thermocycler may comprise other components such mechanics, electronics, firmware and software.

[0061] The amplification of DNA can be done via biochemical reagents which need specific temperatures. Regarding the thermal requirements, some procedures need cycling of different temperature steps, other procedures are isothermal. Typically, the needed temperatures range from 35° C. to 95° C. depending on the application. In general, heating and cooling is necessary. Peltier elements or air may be used for tempering the reagents. There are other methods such as heating via magnetic induction. A very accurate control of the temperature is necessary because the biochemical process is very sensitive to temperatures.

[0062] One way to detect the result of amplification is via fluorescence detection. Fluorescence is generally stimulated by an electromagnetic radiation. The radiation is absorbed by the fluorescent molecule. The fluorescence molecules are also called fluorescence dyes. After absorption the molecule emits radiation which may be a fluorescence signal that can be detected by a detector. The emitted signal is preferably in a different wavelength range than the excitation radiation. Therefore the excitation and emission radiation can be distinguished. Exposing a sample to an excitation radiation and detecting the emitted radiation is part of the field of fluorometry which is known to the art.

[0063] The optical unit for the measurement of signals uses light source for excitation and a detector for detection of the fluorescence signals. Special optical components, such as an optical filter, can be used to provide light of the appropriate wavelength for excitation and detection and to distinguish between the excitation light and the signal. Each fluorescence dye has a specific spectrum. In one well might be several different fluorescence dyes for different targets. That is why different dyes must be excited and detected separately. Typically, there are one to four different fluorescence dyes per well but there can be more. There can be one or several excitation sources and detectors. Suitable light sources can be for example LEDs, laser, halogen lamps or the like. Examples for detectors are photodiodes, photomultiplier, charge-coupled devices (CCD) and others. Mechanical and optical components are needed to bring the excitation light to the reagents and the signal to the detector.

[0064] There are several possibilities for excitation and detection principles such as illuminating and detecting the whole plate, scanning of the wells, one source and detector for each well, bringing the tubes to the optical system and others.

[0065] The measured fluorescence intensity may vary significantly between different biological reagent mixtures and between different technical devices. This variation may lead at the detection unit to poor results or results which are difficult to analyze. For optimum detection, the detected signal should be in the middle of the working range of the detection unit. Most detectors are not linear but have a specific or preferred working range. If the to-be-detected signal is too high the detector may saturate. A too low signal may be very noisy. The optical units, installed in existing real-time cyclers, are very diverse, as described above. Each optical unit has a unique working range, some are very sensitive, others can detect even high fluorescence signals. This diversity has a huge influence on the detected samples. There are some samples which are clearly detected in one system, but are very low and noisy in another one. The other way around, high fluorescence signals may be saturated in one system and correctly detected in another one.

[0066] Regarding the reaction mixtures for the biochemical reactions, they are very diverse in their composition and the concentration of their ingredients. Therefore, the intensity of the fluorescence signal itself may vary considerably. The intensity of the baseline fluorescence and of the end fluorescence in each individual well for each individual dye can be different depending on the properties of the reagent mixture in the well. To adapt the reagent mixture to get higher or lower fluorescence signals is very complex or sometimes not possible.

[0067] In most real-time cyclers a fixed setting is used for the intensity of the excitation source and for the sensitivity of the detector. To detect all signals clearly, a detector must have a very broad detection range. Still the analysis might be difficult because the intensities of the results vary a lot.

[0068] To compensate for different heights of fluorescence signal intensities, the real-time thermocycler can have the possibility to change the settings of the excitation source or the detector. The detector can for example be adapted by changing the gain of a photomultiplier or the integration time of a camera, to bring the fluorescence signal within the correct range of the detector. In this case the fluorescence signal is modified after its generation. This helps preventing saturated signals or to increase the intensity of very low and noisy signals, but these changes affect all samples in one run equally. The potentially huge differences in fluorescence signal intensity between individual samples will nevertheless remain. Therefore, the applied settings are a compromise to generate a best fit in terms of fluorescence signal intensity over all samples, rather than improving each signal individually.

[0069] Prior art real-time thermocyclers face the challenge of variable fluorescence intensities between different targets. This can be overcome by adapting the excitation intensity for each well and fluorescence dye individually, thus allowing optimal signal improvement for low and high signals. This way all signals can be adjusted to be within a preferred range, e.g. a linear range, of the detector and the differences in signal intensities will be reduced. This simplifies the analysis of the resulting amplification curves.

[0070] Preferably, an excitation source is used which is able to adjust its excitation intensity in the range of milliseconds. For the detector no change in sensitivity or other settings is necessary, because the signal intensities are adjusted by the excitation. To handle the settings, control the excitation source and the detector and to manage the data from the optical unit suitable electronic elements can be used.

[0071] To prevent that the user has enter value of the excitation intensity for every dye in every well, the excitation intensity can be automatically chosen, depending on the used target. The user enters the target for a well, and the parameter for the optics are automatically chosen from a database.

[0072] Thus, before a new reagent mixture can be measured automatically, the correct settings are provided to the system. The correct excitation intensity for a reaction mixture can be determined once by measurement and then stored in the database for future use.

[0073] FIG. 1 is a schematic illustration of a real-time thermocycler 100. The real-time thermocycler 100 comprises an optical unit 110, a well 120, a thermal unit 130, and a controller 140. The optical unit 110 comprises an excitation unit 112 and a detection unit 114. The excitation unit 112 is configured to excite fluorescence molecules of a sample in the well 120 through radiation 122. The detection unit 114 is configured to detect a fluorescence signal 124 from the sample in the well 120.

[0074] The thermal unit 130 is arranged next to the well 120 and configured to adjust a temperature of the sample in the well 120. The optical unit 110 is connected to a controller 140. The controller 140 is configured to control the excitation unit 112 to adjust an intensity of the excitation of the fluorescence molecules of the sample based on information about the target.

[0075] The real-time thermocycler 100 optionally (as indicated by dashed lines in FIG. 1) comprises a database 150 and/or a user interface 160. The database 150 may comprise information about the sample. The user interface 160 may be configured to let a user enter the information about the sample.

[0076] FIG. 2 is an illustration of a further real-time thermocycler 200. The real-time thermocycler 200 may be the real-time thermocycler 100 shown in FIG. 1, but only some components are shown in FIG. 2. The real-time thermocycler 200 comprises a first optical unit 210a, which is configured to send a strong excitation signal 222a to a sample in the well 230a. The optical unit 210a measures a strong fluorescence signal 224a from the sample. The real-time thermocycler 200 further comprises a second optical unit 210b, which is configured to send a weaker excitation signal 222b to a second sample in the second well 230b. Since the second sample has stronger fluorescence properties, a second fluorescence signal 224b, which is similar in strength to the first fluorescence signal 224a is emitted from the sample and measured at the optical unit 210b.

[0077] FIGS. 3a and 3b illustrate experimental results obtained with a real-time thermocycler in accordance with the present invention.

[0078] A PCR run with 45 cycles was performed. Three different targets were used for the experiment. The targets were in separate wells but they all used the same fluorescent dye. Several replicates were done which are very similar. Due to clarity, only one curve per target is shown in the graphs in FIG. 3a and FIG. 3b. All targets are measured with the same power of the excitation source. Furthermore replicates of target three are measured with a four times higher excitation power.

[0079] In the graphs the fluorescence signals over the amplification cycles are shown. The single measurement points are linked to form a curve. The curves from target 1 are labeled with 1 in the graph. For target 2 and target 3 it is done likewise but the curves with higher excitation power are labeled with 3a.

[0080] In FIG. 3a and FIG. 3b raw fluorescence signals are shown. The variability of the different curves are explicit: Curve 1 has a high baseline fluorescence and a high end fluorescence. Curve 2 shows a low baseline fluorescence, but a high end fluorescence whereas for curve number 3 baseline and end fluorescence are low. Curve 3a has a high baseline and end fluorescence.

[0081] In comparison to curve 3 the curve 3a has a much higher fluorescence intensity, which results from the higher excitation power. Regarding the detector, curve 3a is in the middle range, whereas curve 3 is in a very low range. The whole detector range is from 0 to 4000. The signals of target 2 reach from a low to a high fluorescence intensity. They can also be optimized regarding the range of the detector.

[0082] For analysis usually the baseline is subtracted to normalize the curves. This allows comparing the different curves regarding the point of rising of the signal. In FIG. 3b the data from FIG. 3a are shown with normalized baseline. The end fluorescence intensities of curves 1, 2 and 3a are in a similar range, whereas the end fluorescence of curve 3 is much lower. Regarding this example it is clearly visible that it is easier to analyze the curves if they have a comparable height especially if a threshold is used.