To develop a small thermal cycler that is easier to use, easier to carry, and less expensive than traditional systems.
Use NI’s CompactDAQ hardware system and LabVIEW software to create a small thermal cycler and perform real-time PCR with the plug-and-play capabilities of the USB interface.
Hsieh Tseng Ming James – Institute of Bioengineering and Nanotechnology, A-Star
Polymerase chain reaction (PCR) thermal cycling is the gold standard for molecular diagnostics. However, the main challenge facing the world in responding to the pandemic is that only professionals can perform effective diagnostic tests. In addition, commercially available thermal cyclers can only be used in laboratory environments, so it is difficult for us to operate them. To expand the application field of commercial thermal cyclers to emergency situations, as well as public testing stations, such as Airports, with their bulky and expensive nature, are certainly a hindrance. Therefore, there is an urgent need for us to develop an inexpensive, portable, and disposable molecular diagnostic tool.
In order to provide a flexible, low-cost diagnostic system, our research group at the Singapore Institute of Bioengineering and Nanotechnology has developed a small diagnostic system using PCR technology recommended by the US Centers for Disease Control and Prevention (CDC) to Detection of infectious emergencies. Our system integrates and automates the entire diagnostic process, from real-time PCR to detection of viral gene target strands, to perform data analysis.
By using National Instruments’ CompactDAQ hardware system and LabVIEW graphical system design software for control and data analysis as our solution, we created a small and portable system. The system consists of a small Peltier device (temperature device), power supply (energy device), and a photodetector (optical device) with USB plug-and-play functionality.
Our thermal cyclers are portable, fully automated, and can be used in the context of large-scale health checks in critical situations, such as controlling the spread of infectious diseases at airport checkpoints. System automation is a key advantage for reliable diagnostics, as influenza pandemics can occur in remote areas where there may not be professionally trained personnel handling test samples. In addition, the realization of system automation greatly reduces manpower and training costs.
Using this system to run three polymers containing preloaded PCR reactions simultaneously takes less than an hour from sampling to result and allows for the use of a large biological diversity. Using real-time temperature feedback from thermistors, an internal proportional-integral-derivative (PID) chip handles temperature control. The controller consists of a compensator, an output device and a sensor feedback signal, which is necessary to control the heater output power and maintain temperature. Using LabVIEW software, we programmed the graphical user interface to Display real-time temperature, set temperature, and Display real-time PCR results from three channels (as shown in Figure 1A).
The configuration of the entire integrated system includes a cyclic temperature control system to perform PCR system operation and reactions, three blue LED light sources fixedly mounted, and associated lenses for real-time optical detection between the LED and the photomultiplier tube (PMT) optical path and filter. The system includes a set of copper or brass heating chambers integrated with the polymer reaction chamber where the sample is placed.
LabVIEW is at the heart of the system architecture, and the central processor unit is designed to execute instructions pre-written in LabVIEW to control system startup and health checks, thermal cycling control of PCR runs, and optical detection of multiplexed fluorescent signals.
During the cyclic temperature control of PCR processing, the system performs optical detection in the last second of the annealing cycle. The three LEDs are simultaneously lit sequentially by current from the NI 9265 analog output module. Each LED is lit continuously for 200 ms, and the current LED is turned off before the next LED is lit.
A specially designed device is used to focus and guide the LED light path transmission. The light passes through a filter before hitting the reaction chamber where the DNA sample is placed. The light passes through a series of lenses and filters before reaching the PMT, and the signal is finally read out by the NI 9206 analog input module.
We used LabVIEW for signal processing to obtain the mean of the dataset, and the signal from the PMT was amplified and processed. The data is then displayed to the user and continuously updated in three separate graphs via the main user menu (see Figure 2).
The PCR mix was placed in a polycarbonate PCR reaction chamber and oiled to prevent evaporation. PCR was performed under the following temperature conditions: 95 °C for 20 s (Taq polymerase activation), 50 cycles of amplification (5 s at 95 °C for heat denaturation, 60 s at 60 °C for annealing and extend). The LabVIEW program records the fluorescence emitted by DNA replication during each cycle.
Stability and Reusability
In molecular diagnostics, PCR is a common method for amplifying specific DNA fragments to the molecular level. The realization process required 40 to 50 cycles of thermal transformation at 94 °C, annealing at 60 °C, and elongation at 60 °C. The key point of the whole process is precise temperature control, maintaining a temperature rise rate of 2 °C/s to avoid generating or amplifying erroneous DNA sequences.
We used LabVIEW to control independent high-side and low-side reference output channels, and designed and fabricated an on-chip heating scheme using a circuit consisting of high-speed metal-oxide-semiconductor field-effect transistors (MOSFETs), enabling real-time PWM control. Temperature measurements showed that we obtained heating and cooling rates of 2.5 °C/s and 2.2 °C/s, respectively. Overshoot is less than 1 °C and stability is maintained at ±0.1 °C for each temperature setting.
Results and Conclusions
We confirmed the real-time detection performance of PCR by serial dilutions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNA (cDNA) using TaqMan probes ranging from 1 to 104. The results confirmed that the sensitivity determined from the Ct curve was comparable to the performance of a real-time thermal cycler on the market (Fig. 1B).
Compared to other thermal cyclers on the market, our system is more compact, more portable, and better constant temperature (Table 1). The portability of the system allows it to play a greater role in the early stages of a widespread flu outbreak. At the same time, the diagnostic system is very suitable for use in outpatient clinics, emergency rooms, public inspection sites, etc. In addition, the thermal cycler has potential advantages in food safety due to its good portability, low price, small size, and ease of use.
1. J. Felbel, I. Bieber, JM Kohler, Chemical Surface Management for Micro PCR in Silicon Chip Thermocyclers, Proc. SPIE, 4937, 34-40, 2002.
2. S. Poser, T. Schulz, U. Dillner, V. Baier, JM Kohler, G. Mayer, A. Siebert, D. Schimkat, Temperature Controlled Chip Reactor for Rapid PCR, 2002.
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