NEW YORK (GenomeWeb) – Vintage biotech methods aren't often revived, but the old-fashioned PCR technique of dunking tubes in baths of heated water has now been adapted, with a few tweaks, into an inexpensive thermal cycler for use in low-resource settings.
In a proof-of-principle study published this week in PLoS One, researchers at College Station, Texas-based AI Biosciences have automated the archaic PCR method, showing setups built from Thermoses, soup cans, and microcontrollers, can rival the efficiency of commercial platforms.
The method taps into the unique thermal properties of water, but also has an innovative way to prevent heat loss, Season Wong, AI Biosciences' co-Founder and president, told GenomeWeb in an interview.
Most modern thermal cyclers use metal heating blocks. Some companies are trying to make PCR faster by optimizing the thermal mass of these blocks, for example by using thin aluminum plates, as recently reported. Others drive speeds faster by using high concentrations of reagents, special polymerases, or lasers, which may all prove too expensive for the developing world.
On the other hand, to drive down costs some newly described methods use isothermal nucleic acid amplification and single heat sources, such as exothermic chemical reactions or even body heat. But these techniques usually require that established molecular diagnostics be redesigned.
Microfluidic PCR devices can be constructed for spatial thermal cycling, but have drawbacks in terms of cost and loss of small sample volumes to the inner surfaces of the chamber.
So, to do traditional PCR on the cheap, it ultimately boils down to needing a thermal cycler, and these usually cost at least a few thousand dollars.
Wong said he reasoned that if heat loss could be prevented in an inexpensive way, the old-fashioned water bath PCR might actually best meet all the requirements of low-resource settings.
The device described in the PLoS paper, which costs about $130 to build and can be as fast as 17 seconds per cycle, specifically uses Thermos brand vacuum containers because they are readily available. Wong said the group tried other brands in an attempt to drive costs further down, but those did not maintain the temperature as well.
But the innovation that enables the technology is ultimately a layer of cooking oil on the surface of the water. This prevents heat from escaping in the form of steam.
"The oil is the trick," Wong said.
In the study, the oil allowed water initially at about 96 degrees to maintain temperature above 90 degrees for 60 minutes, giving plenty of time for hot-starting the polymerase and cycling through the DNA denaturation dunks.
The method can also accommodate a variety of different styles of PCR tubes, and it was shown effective with LED and cell-phone based detection.
Although a laptop and some small expertise is required to program the commercially available Arduino-based microcontroller, the only power source besides batteries required after set-up is whatever it takes to boil the water.
The temperature in the denaturation bath is maintained above 90 degrees by pouring boiling water into the Thermos containing 11 milliliters of oil. A mathematical equation describes the proportion of room temperature water that must be added to boiling water to achieve any desired annealing and extension bath temperature in a second Thermos.
The microcontroller is then mounted onto a soup can, and wire and a stick are fashioned into a dunking arm.
AI Biosciences, which is currently funded via US National Institutes of Health Small Business Innovation Research Grants, will ultimately combine this thermal cycler with previously described sample prep using a multichamber preloaded syringe and other modular technologies.
"Our passion is to develop point-of-care molecular devices for low-resource settings," Wong said.
The firm is also working on novel detection methods and a real-time method using the Thermos set-up. The technique can also be easily adapted to run RT-PCR.
The company hasn't finalized any plans to make the Thermos thermal cycler available as a DIY kit, and it is open to potential future collaborations or investment, Wong said.