WO2015189297A1

WO2015189297A1 - Method for operating a real-time polymerase chain reaction (pcr) system and a device for operating the method

Info

Publication number: WO2015189297A1
Application number: PCT/EP2015/062986
Authority: WO
Inventors: Pavel NEUŽIL
Original assignee: Kist Europe-Korea Institute of Science and Technologie Europe Forschungsgesellschaft mbh
Priority date: 2014-06-10
Filing date: 2015-06-10
Publication date: 2015-12-17
Also published as: DE102014108144B4; DE102014108144A1

Abstract

The present invention relates to a method for operating a real-time polymerase chain reaction (PCR) system and a device for operating the method. It is the object of the invention to provide a new method, which allows the manufacture of a small and portable real-time PCR system. The object is met by the method for operating a real-time polymerase chain reaction system comprising a control system for temperature regulation of a sample holder, whereby this regulation is of the closed-loop type, for at least two temperature set values, whereby at a first time the closed-loop temperature regulation is started and at a second time, which is after the given first time, the measurement of the fluorescence intensity and/or luminescence decay time starts, whereby at the second time the components of the control system required for the closed-loop temperature regulation are at least partly used for the fluorescence intensity and/or luminescence decay time measurements, while at the second time the closed-loop temperature regulation is interrupted for the measuring period.

Description

Method for operating a real-time polymerase chain reaction (PCR) system and a device for operating the method.

The present invention relates to a method for operating a real-time polymerase chain reaction (PCR) system and a device for operating the method.

PCR is a technology to amplify copies of specific deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) fragments in a reaction chamber. Due to simple and easy application using repeated cycles of three steps, namely denaturation, annealing and extension, the PCR technology has been proven to be highly efficient and reliable. Hence, this PCR technology has been extensively used in medicine, science, agriculture, veterinary medicine, food science, environmental science as well as in molecular biology, archaeology and anthropology.

In recent years genetic analysis also became a commonly used technique for detecting the presence and potential for disease. Blood or tissue samples, taken in a minimally invasive manner from localized regions, can be used to perform DNA analysis via PCR. For quantification, generally, the end-point PCR products are separated on a gel and the approximate amount is estimated using spectrophotometers. However, an accurate measurement of the amount of DNA is not achievable with this end-point method. Thus, to overcome these problems, a real-time PCR method and a fluorescence detection method have been introduced in which at each PCR cycle the accumulated PCR products are measured. Using these two methods an amplification plot showing fluorescent intensities versus cycle numbers can be obtained.

However, most of the analysis is carried out in laboratories, which use systems that are designed for high-throughput analysis. The samples taken in the field are sent to these laboratories, where the DNA is extracted and placed in a chamber with a so called PCR cocktail, whereupon the analysis is run.

Having an easily portable real-time PCR instrument to immediately perform the DNA analysis in the field is thus extremely valuable. A few real-time PCR devices have been described which comprise an integrated fluorescence detection unit.

Belgrader et al. (P. Belgrader, S. Young, B. Yuan, M. Primeau, L.A. Christel, F. Pourahmadi, and M.A. Northrup, Anal. Chem. 73, 286-289 (2001)) developed a compact, real time PCR instrument for rapid, multiplex analysis of nucleic acids in a portable format by using a thin- filmed resistive heater, a fan, an LED and silicon photodiode detectors. The instrument weighs 3.3 kg, measures 26 x 22 x 7.5 cm and can run continuously on the internal batteries for 4 hours.

Even though this system is usable as a portable real-time PCR instrument, the reaction mixture needed is between 25 and 100 reaction volume and the dimensions and the weight are rather unhandy.

Higginns et al. (J.A. Higgins, S. Nasarabadi, J.S. Karns, D.R. Shelton, M. Cooper, A. Gbakima, and R.P. Koopman, Biosens. Bioelectron. 18, 1115- 1123 (2003) reported on the use of a novel handheld nucleic acid analyzer instrument, weighing less than a kilogram and capable of performing four real-time PCR reactions by using plastic PCR tubes with a reaction volume of 25 - 30 μί.

Da-Sheng Lee et al. (D.S. Lee, M.H. Wu, U. Ramesh, C.W. Lin, T.M. Lee, and P.H. Chen, Actuat. B-Chem. 100, 401-410 (2004b)) developed a miniature spectrometer for detecting the emission of fluorescence intensity from RT-PCR mix in a micro liter volume glass capillary.

Even though they developed a miniature spectrometer, the dimensions of the system are the ones of a commercial thermal cycler and hence not portable.

Xiang et al. (Q. Xiang, B. Xu, R. Fu, and D. Li, Biomcd. Microdev. 7, 273-279 (2005 )) developed an on-chip real time PCR device using a miniature thermal cycler, however, realtime detection is performed by using a desktop fluorescent microscope and thus this system is not portable. Xiang et al. (Q. Xiang, B. Xu, R. Fu, and D. Li, Biomed. Microdev. (2007)) also developed a chip based real-time PCR system comprising a PDMS reactor chip, miniaturized thermo- cycler and a fiber optical fluorescence excitation and detection module. However, this system is not portable.

It would therefore be of great benefit if a device for the direct quantification of PCR products could be built, which overcomes or alleviates one or more of the above problems.

It is the object of the invention to provide a new method, which allows the manufacture of a small and portable real-time PCR system.

The object is met by the method for operating a real-time polymerase chain reaction (PCR) system comprising a control system for temperature regulation of a sample holder, whereby this regulation is of the closed-loop type, for at least two temperature set values, whereby at a first time the closed-loop temperature regulation is started and at a second time, which is after the given first time, the measurement of the fluorescence intensity and/or luminescence decay time starts, whereby at the second time the components of the control system required for the closed-loop temperature regulation are at least partly used for the fluorescence intensity and/or luminescence decay time measurements, while at the second time the closed-loop temperature regulation is interrupted for the measuring period.

Thus a miniaturization of the real-time PCR system can be achieved, since only one control block, which may be a single-lock in amplifier, for both, temperature regulation of the closed- loop type and the real-time PCR product signal measurement, is present. The PCR product signal may be fiuorescence signal emitted from the PCR chamber, measured preferably as fluorescence intensity or luminescence decay time. However, the PCR product signal may also be measured applying absorption detection strategies.

Usually in lab-positioned high throughput PCR systems the temperature maintenance in the sample wells is dependent on the thermoblocks in which said samples are placed. In these thermoblocks since they have a low thermal conductivity the heat is generally maintained during the measurement period. Thus there is no temperature control of the open-loop type demanded. However, small chips such as microfluidic chips composed of silicone, glass, metal or plastics which can be applied for the PCR have a high thermal conductivity and therefore easily transfer heat.

Hence, it is within the scope of the invented method that the temperature is controlled during the measurement period by a temperature regulation of the open-loop type. This open-loop regulation of the temperature has the advantage that only a small number of components are required. Therefore the required space for the open-loop control system in the device can be reduced.

It is envisaged that the correcting variables for the open-loop temperature regulation are determined dependent on the correcting variables between the first and the second time during the closed-loop regulation.

Hereby the correcting variables for the open-loop temperature regulation can be determined dependent on averaged values of these correcting variables, which were evaluated during a time period starting before the second time and under the condition that the maximum amount of deviation of the measured temperature to the defined temperature set value is smaller than a set threshold, and ending at the second time.

Hereby, the influence of the environment on the temperature, as evaluated in the period of the closed-loop regulation, can be taken into account during the period of the open-loop regulation.

This allows the temperature to be controlled via the average duty cycle during the PCR product signal emission and recording.

Hereby, it is advantageous that the temperature is at least nearly maintained at the set value. Even though thermoblocks have a low thermal conductivity, the temperature on the outer wells may be affected differently than the temperature for the wells in the core. This may lead to measurement differences and thus to errors. However, by applying an open- loop temperature regulation, temperature differences for the at least one sample well can be avoided. It is further a preferred embodiment of the invention that the open-loop temperature regulation is at least partly handled via components, which are used in the time period from the first to the second time for the closed-loop temperature regulation and which are not used for the fluorescence intensity and/or luminescence decay time measurements starting at the second time.

This multiplexing of closed-loop temperature regulation and fluorescence or luminescence decay time measurements is advantageous. During PCR operation, meaning the denaturation, annealing and until the second time the extension step, the system operates with a closed-loop temperature regulation with preferably pulse width modulation (PWM) of the power for the heater. During this time period the fluorescence or luminescence measurements are not executed. The time point, from which on the samples are measured, has been here assigned as the second time. Usually the measurements during PCR cycling are taken at the end of the primer extension/elongation period. In the case of using Thermus aquaticus (Taq) polymerase the fluorescence is commonly measured at 72 °C. However, for the measurements also other DNA polymerases, having an optimum activity temperature around 70 °C may be used.

It is feasible within the scope of the invention that the measurement is performed by means of multiple detectors, the detectors being controlled individually and subsequently during the measurements.

This is advantageous, because even though only one detector can detect a number of tubes by using optical fibers, this requires an expensive light source which has a good coherency, such as a laser, to collect an excitation beam for exciting fluorescence on optical fibers.

However, for the preferred embodiment the detection unit may be a single-channeled or multi-channeled fluorescent detector that detects the emitted fluorescent intensity. The detector could also be a charge-coupled device (CCD) camera or sensor with optical fibers, a photodiode or a photomultiplier tube (PMT). In a preferred embodiment it is money- wise expedient that the PCR system utilizes a PMT in addition or preferentially instead of a CCD camera. Hence the PCR product signal may be an electrical signal to which an alternating current is applied and the detection unit may comprise a sensor that detects the signal. Typical real-time quantitative PCR systems comprise a laser to induce fluorescence and in order to read from all samples in a plate, the plate is moved, whereby the laser individually addresses each and every well in the plate. However, a system such as this is large and expensive.

To avoid above mentioned drawbacks it is thus advantageous that the measurement is performed by means of multiple transmitters, whereby the transmitters may be controlled individually and subsequently during the measurements.

Hereby the transmitter can be any light source that causes fluorophore excitation, including for instance lasers, photodiodes, laser diodes and lamps such as blue light emitting diodes (LEDs). However, it is advantageous to have a transmitter which is small, reliable and able to excite the dye, such as SYBR^® Green I, preferably used in the method. It is further advantageous that the transmitters are inexpensive. Thus blue light emitting diodes (LEDs) or laser diodes are a preferred choice for the transmitter.

Because the footprint of an LED or laser diode is very small, it is further conceivable that multiple LEDs or lasers of different wavelength could be integrated into a single package or several packaged LEDs/laser, which can be very closely spaced to excite one well/sample.

It is further within the scope of the invented method that the at least one transmitter is only activated under the condition that the closed-loop temperature regulation is inactive.

This is advantageous because fluorescent dyes used for the measurement are light sensitive and therefore subject to photo bleaching. In the invented method the samples are only exposed for the time necessary to obtain the data.

A device for carrying out the above mentioned method, wherein the components of the closed-loop control system comprise at least one lock-in amplifier, whereby first input signal means are assigned to the lock-in amplifier for connecting temperature measurement means, whereby further input signal means are assigned to the lock-in amplifier for connecting at least one optical detector, whereby furthermore output signal means are assigned to the lock- in amplifier for connecting at least one heating device is also within the scope of the invention.

It is of advantage that the electronics needed for measurement and temperature regulation are performed by the same lock-in amplifier based electronics. This allows a simplified control system architecture and consequently miniaturization. The result of the miniaturization may be a handheld device or even be a pocket-sized device, both which can be easily carried around.

Hence, it is within the scope of the invention that the device has smaller dimensions than 7.0 cm x 12.0 cm x 19.0 cm and weighs less than 0.5 kg. However, it is also within the scope of the invention that the device has equal or smaller dimensions than 3.5 cm x 6.0 cm x 9.5 cm and weighs less than 0.2 kg.

The above mentioned features which allow miniaturization further allow for a smaller reaction volume.

It is thus within the scope of the invention that the solution volume (reaction volume) is less than 1.5 μΐ,. However, it is also feasible to use solution volumes in the range of 0.1 μΙ_, to 1.0

It is further advantageous and envisaged that the system is accumulator- or battery-powered.

An instrument which enables genetic analysis in the field by providing a handheld device is of interest for field research studies, studies in remote areas and temporary laboratories. The device may include a data acquisition computer for analyzing the reactions in real-time, or the device may communicate the data to another device through wired or wireless communication interfaces. In addition, the device which is described for the invention may be paired with other technologies, such as capillary gel electrophoresis, mass spectrometry or sequencing for further analysis of the samples. Another possibility of applying such an instrument may be to rapidly detect or identify people or the presence of disease, with application in military or the civilian sector. Such an instrument can be further used, for example, at airports for identification of wanted individuals. Another possible application is to detect virus infection or other diseases on people arriving from outbreak regions. In general, this portable device can help to identify potential and existing medical disorders directly on site.

The above and other features, details and advantages of the present invention will become more apparent by describing in detail an exemplary embodiment of the method, which is shown in Fig 1 depicting:

FIG. 1 shows a timing sequence of the method for regulation of the temperature, followed by fluorescence intensity and/or luminescence decay time measurements,

Fig. 2 shows a portable device (system) for operating a real-time polymerase chain reaction.

The invented method applies multiplexed control, meaning, there is only one control block which is used for both, temperature as well as fluorescence intensity and/or luminescence decay time measurement. It is envisaged that multiple (meaning a multiple of 2) fluorescence or luminescence measurements can be performed using the proposed multiplexed control method. It is envisaged that all fluorescent measurement is performed by the same lock-in amplifier based electronics and hence, there is no need to have the identical electronics multiple times. This feature allows among others for very compact electronics, an important feature for a portable real-time PCR device. In addition, the temperature measurement is also performed by the same lock-in amplifier based electronics (lock-in amplification). Thus only one single lock-in amplifier is used to either measure the temperature or the fluorescence intensity and/or luminescence decay time.

One proposed sequence for measuring the temperature followed by the measurement of here the fluorescence is depicted in Fig. 1, which shows a device which enables the fluorescence measurement of four samples. As shown in Fig. 1 A, for the time span from n to n+8, whereby n can be any time point during normal PCR operation, the fluorescent measurement is turned off (OFF, meaning also, that no fiuorophore excitation by lasers, photodiodes, laser diodes and lamps such as blue light emitting diodes (LEDs) takes place) and the single lock in amplifier is used to measure the temperature (ON). The system operates with a closed- loop temperature regulation with preferably pulse width modulation (PWM) of the power for the heater, thus as soon as the second time point is reached (here time point n+8, Fig. 1A) the temperature measurement is stopped to be executed (OFF), however the temperature can be maintained at a predefined level by using average duty cycling. Generally during PCR cycling, the fluorescence is measured at the end of the of the primer extension/elongation period. Thus, n can e.g. be the time point which indicates the last ten seconds of the extension/elongation period. Since a system is shown which allows measurement of four samples, the fluorescence is measured sequentially in the four probes. For this, four times fluorophore excitation, preferably by blue light emitting diodes (LEDs), is activated sequentially; each for approximately 0.5 seconds (Fig. IB to Fig IE) and the fluorescence is measured. In the Fig. IB to Fig IE the measurement of the fluorescence intensity and/or luminescence decay time is turned ON at time point n+8 (second time).

Fig. 2 shows the inventive portable PCR device. The device has dimensions of maximum 3.5 cm x 6.0 cm x 9.5 cm (width x height x length) and weighs less than 0.2 kg. It is thus feasible to carry the device in one hand in the field, as e.g. needed for field research studies, studies in remote areas or for temporary laboratories. The features of the invention further allow for a reaction volume in the range of 0.1 to 1.0 μί.

Claims

Method for operating a real-time polymerase chain reaction (PCR) system comprising a control system for temperature regulation of a sample holder, whereby this regulation is of the closed-loop type, for at least two temperature set values, whereby at a first time the closed-loop temperature regulation is started and at a second time, which is after the given first time, the measurement of the fluorescence intensity and/or luminescence decay time starts,

characterized in that at the second time the components of the control system required for the closed-loop temperature regulation are at least partly used for the fluorescence intensity and/or luminescence decay time measurements, while at the second time the closed-loop temperature regulation is interrupted for the measuring period.

Method according to claim 1 ,

characterized in that the temperature is controlled during the measurement period by a temperature regulation of the open-loop type.

Method according to claim 2,

characterized in that the correcting variables for the open-loop temperature regulation, are determined dependent on the correcting variables between the first and the second time during the closed-loop regulation.

Method according to claim 3,

characterized in that the correcting variables for the open-loop temperature regulation are determined dependent on averaged values of these correcting variables, which were evaluated during a time period starting before the second time, under the condition that the maximum amount of deviation of the measured temperature to the defined temperature set value is smaller than a set threshold, and ending at the second time.

Method according to claim 1 to 4,

characterized in that the open-loop temperature regulation is at least partly handled via components, which are used in the time period from the first to the second time for the closed-loop temperature regulation and which are not used for the fluorescence intensity and/or luminescence decay time measurements starting at the second time.

6. Method according to claim 1 to 5,

characterized in that the measurement is performed by means of multiple detectors, the detectors being controlled individually and subsequently during the measurements.

7. Method according to claim 1 to 6,

characterized in that the measurement is performed by means of multiple transmitters, the transmitters being controlled individually and subsequently during the measurements.

8. Method according to claim 1 to 7,

characterized in that the at least one transmitter is only activated under the condition that the closed-loop temperature regulation is inactive.

9. Device for carrying out the method according to at least one of the preceding claims, characterized in that the components of the closed-loop control system comprise at least one lock-in amplifier, whereby first input signal means are assigned to the lock-in amplifier for connecting temperature measurement means to the lock-in amplifier, whereby further input signal means are assigned to the lock-in amplifier for connecting at least one optical detector, whereby furthermore output signal means are assigned to the lock-in amplifier for connecting at least one heating device.