Developing a smarter portable influenza diagnostic system

"Using NI's Compact DAQ hardware system and LabVIEW graphical system design software for control and data analysis, we created a small and portable system."

– Hsieh Tseng Ming James, Institute of Bioengineering and Nanotechnology, A-Star

The Challenge:
Develop a small thermal cycler that is easier to use, portable, and less expensive than traditional systems.

The Solution:
Use NI's CompactDAQ hardware system and LabVIEW software to create a small thermal cycler that can perform real-time polymerase chain reaction using the plug-and-play capabilities of the USB interface.

Polymerase chain reaction (PCR) thermal cycling is the gold standard for molecular diagnostics. However, a major challenge facing the world in responding to the influenza pandemic is that only professionals can perform effective diagnostic tests. In addition, commercially available thermal cyclers are only for use in laboratory settings, making them difficult to operate, and their bulky and expensive nature is undoubtedly an obstacle to expanding the application of commercial thermal cyclers to emergency situations and public testing stations, such as airports. Therefore, it is urgent to develop a cheap, portable, and disposable molecular diagnostic tool.

To provide a flexible and low-cost diagnostic system, our research team at the Singapore Institute of Bioengineering and Nanotechnology has developed a small diagnostic system that uses the PCR technology recommended by the Centers for Disease Control and Prevention (CDC) to detect infectious diseases. Our system integrates and automates the entire diagnostic process from real-time PCR to the detection of viral gene target strands to perform data analysis.

By using NI's CompactDAQ hardware system and LabVIEW graphical system design software for control and data analysis as our solution, we created a small and portable system consisting of a small Peltier device (temperature device), power supply (energy device), and light detector (optical device) with USB interface plug-and-play function.

Our thermal cyclers are portable, fully automated, and can be used in critical mass health screening situations, such as at airport checkpoints to control the spread of infectious diseases. System automation is a key advantage for reliable diagnostics, as influenza pandemics may occur in remote areas where there may not be trained personnel to handle test samples. In addition, the implementation of system automation significantly reduces manpower and training costs.

The system was used to run three polymerases simultaneously containing preloaded PCR reactions, with a sampling to result time of less than an hour, and allowed the use of a wide variety of organisms. An internal proportional integral derivative (PID) chip handles temperature control using real-time temperature feedback from thermistors. The controller consists of a compensator, output device, and sensor feedback signals, which are necessary to control the heater output power and maintain temperature. Using LabVIEW software, we programmed a graphical user interface to display real-time temperature, set temperature, and display real-time PCR results from three channels (as shown in Figure 1A).

System Configuration
The configuration of the entire integrated system includes a circulating temperature control system for executing PCR system operation and reaction, three fixed blue LED light sources, and related lenses and filters arranged between the LED and photomultiplier tube (PMT) optical path for real-time optical detection. The system includes a set of copper or brass heating chambers integrated with the polymer reaction chamber where the sample is placed.

LabVIEW is the core of the system architecture. The central processor unit is designed specifically to execute instructions pre-written by LabVIEW. It can be used to control system startup and health checks, thermal cycle control of PCR runs, and optical detection of multiplexed fluorescence signals.

During the temperature control cycle of the PCR process, the system performs optical detection at the last second of the annealing cycle. The three LEDs are simultaneously driven by the current from the NI 9265 analog output module and light up in sequence. Each LED lights up for 200 ms and turns off before the next LED lights up.

A specially designed device is used to focus and direct the LED light path. The light passes through a filter before it hits the reaction chamber where the DNA sample is placed. The light also passes through a series of lenses and filters before reaching the PMT, where the signal is read by the NI 9206 analog input module.

We use LabVIEW to perform signal processing to obtain the mean of the data set, and the signal from the PMT is amplified and processed. The data is then displayed to the user and continuously updated in three separate graphs through the main user menu (see Figure 2).

The PCR mixture was placed in a polycarbonate PCR reaction chamber and filled with oil to prevent evaporation. PCR was performed under the following temperature conditions: 95 °C for 20 seconds (activation of Taq polymerase), 50 cycles of amplification (heat denaturation at 95 °C for 5 seconds, annealing and extension at 60 °C for 60 seconds). The LabVIEW program recorded the fluorescence emitted by DNA replication during each cycle.

Stability and reusability
In molecular diagnostics, PCR is a common method to amplify specific DNA fragments to the molecular level. During the implementation process, 40 to 50 cycles of thermal denaturation at 94 °C, annealing at 60 °C, and extension at 60 °C are required. 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 incorrect DNA sequences.

We used LabVIEW to control the independent high-side and low-side reference output channels, and designed and fabricated an on-chip heating solution using a circuit composed of high-speed metal-oxide semiconductor field-effect transistors (MOSFETs) to achieve real-time PWM control. Temperature measurements showed that we achieved heating and cooling rates of 2.5 °C/s and 2.2 °C/s, respectively. For each temperature setting, the overshoot was less than 1 °C, and the stability was maintained at ±0.1 °C.

Results and Conclusions
We used TaqMan probes ranging from 1 to 104 to confirm the real-time detection performance of PCR with serial dilutions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNA (cDNA). The results confirmed that the sensitivity determined from the Ct curve was comparable to the performance of commercial real-time thermal cyclers (Figure 1B).

Compared with other thermal cyclers on the market, our system is more compact, more portable, and has better thermostatic properties (Table 1). The portability of the system makes it more useful in the early stages of a large-scale influenza outbreak. At the same time, this diagnostic system is very suitable for use in outpatient clinics, emergency rooms, public inspection sites, etc. In addition, due to its good portability, low price, small size, and ease of use, this thermal cycler has potential advantages in food safety.

References
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.