NEW YORK (GenomeWeb) — Researchers at the University of California, Santa Barbara have developed a new PCR platform for low-resource settings that utilizes heat generated by a personal computer to perform thermocycling, and detects amplification using a mobile phone camera.
The PC-PCR-phone platform, called P3, is designed to amplify nucleic acids directly from blood using capillary tubing loaded with reagents, and, in a recent study, was proven to be able to amplify genomic DNA of Trypanosoma cruzi, the parasite that causes Chagas disease, from whole blood at concentrations four-fold below clinical titer.
The P3 platform was developed in the lab of Tom Soh at UCSB. Faye Walker, an engineering graduate student in Soh's lab, was first author on a paper describing P3 recently published in Analytical Chemistry.
"Anyone who has had the experience of running five data analysis applications, blasting music, and streaming online videos at once will be keenly aware of the waste heat generated by their computer," Walker told PCR Insider in an email this week.
Kareem Ahmad, a Soh lab alumnus and study co-first author who is now a scientist at Illumina, originally had the idea that "the energy expended during computational work could be harnessed for use in the biomedical field," Walker said.
"It only took several minor adjustments to a routine PCR protocol — an initial stepdown phase to account for temperature variances, and DMSO to lower the denaturing temperature of duplexes — to turn his dream of P3 into a reality," she said.
The P3 uses two software programs in tandem, creating a closed-loop feedback to automate the thermal cycling process, Walker said.
With one program, "it is possible to control all the cooling fan units in the PC by linking those fans to temperature monitors provided with the PC hardware. In this case, we use the software to access the internal thermocouple of the CPU [and] based on a set temperature that we input into [the software], the cooling fan for the CPU will automatically be manipulated to perform the cycles of PC-PCR," she said.
The second piece of software maximizes the CPU usage in parallel with the heating steps of the PCR reaction, "so that the temperature is as high as possible within the allowable bounds of CPU operation," Walker said.
The current method utilizes unprocessed blood samples, which do tend to contain PCR inhibitors.
For this reason, the protocol relies on a mutant Taq polymerase, Hemo KlenTaq, from New England Biolabs. "Hemo KlenTaq is resistant to the inhibitors present in whole blood, and is able to tolerate the 5 percent of human blood used in our P3 experiments with T. cruzi genomic DNA, [so] no extra technical steps to extract DNA are required," Walker said.
The platform also uses capillary tubes pre-loaded with PCR reagents — polymerase, oligos, dNTPs, dye, and buffer. "All that is required on the front end is addition of a blood sample to the tubing," she said.
The choice of perfluoroalkoxyl, or PFA, tubing for the capillaries "means that the tubes have a minimal exposure to outside air during the sample prep, are narrow enough to fit within the fins of the heat sink cooling fan for carrying out PC-PCR, and have a low intrinsic fluorescence in the final imaging stage," she added.
The system can run up to 29 samples simultaneously, in its current configuration.
Low-resource settings, however, do not always have reliable sources of electricity. Overcoming reliance on power sources is the next step in development, Walker said. "By carrying out PC-PCR on a laptop instead of a PC, and visualizing end-point fluorescence using blue light instead of a UV transilluminator, we could effectively create a portable, nucleic acid-based diagnostic device."
Walker suggested that the switch to LED visualization would also further lower the cost of equipment.
In addition to migrating the system to a laptop and using LED light for detection, the team intends to demonstrate the platform is more broadly useful for "a vast range of infectious, inherited, and genetic diseases that have been characterized for use in PCR-based assays," Walker said.
Soh's lab had previously developed a platform called Magnetic Integrated Microfluidic Electrochemical Detector, or MIMIED, an aptamer-based technology used to detect influenza H1N1 by RT-PCR. That platform came out of a larger project that was funded by a $3.2 million grant from the National Institutes of Health to develop a system to discover and mass produce high-performance aptamers for use in point-of-care diagnostics and other applications. One of the developers of MIMIED is now CEO at Santa Barbara-based start-up Aptitude Medical Systems, Walker said, so "that technology may very well make an appearance in industry soon."
Regarding the P3 technology, Walker added that "we do not currently have any corporate sponsors or industry partnerships, [but] we invite interested parties to contact us."