NEW YORK (GenomeWeb) – Recombinase polymerase amplification, or RPA, is an isothermal nucleic acid amplification chemistry optimally incubated at 37° C. While this temperature temptingly suggests that even body heat might be used to run RPA reactions, necessary experiments had never been done.
Now, in a proof-of-concept study published earlier this month in PLoS One, researchers at Rice University have demonstrated that incubating an RPA reaction using body heat ― in a cloth pouch tied over the shoulder, with the reaction tube held under the arm ― is sufficient to detect as few as 10 copies of HIV-1 DNA using lateral flow strips.
Co-first authors on the study, Zachary Crannell and Brittany Rohrman, said in an interview that to their knowledge this is the first time anyone has done isothermal PCR with body heat.
"We've seen it anecdotally before, everyone always [says] it would be theoretically possible to perform RPA without a heater, but we hadn't seen anyone do it so we thought it might be an interesting experiment," Crannell said. It had also been suggested that even ambient temperatures are high enough to run RPA in some climates, "but it had all been theoretical in the past," Rohrman added.
In the small study, 10 volunteers heated a small tube of water for 45 minutes, either in their fists, in a rear pants pocket, taped to their abdomens, or held in the underarm, or axilla. After comparing mean temperatures, the researchers found the axilla provided the most consistent heating.
The axilla method involved creating a cloth pouch for the incubation tube, which was then secured over the shoulder and under the arm. The researchers also compared the quality of heating using three different pouch fabrics, including a commonly-available cotton textile Rohrman picked up in a street market while doing a different study in Malawi.
Axilla heating with the cotton fabric was used to test RPA amplification of HIV-1 DNA, but the researchers say the method could also be used for other nucleic acid detection.
Volunteers in the Rice study were other researchers, so they understood that the synthetic HIV target was not infectious, Rohrman said. The study suggested changing the design of the cloth pouch may help alleviate potential concerns about hygiene, should this method be taken into the field. Lysing cells to release pro-viral DNA from whole blood would also make HIV non-infectious, Rohrman noted. The group has tried boiling samples with sodium hydroxide as one inexpensive technique for this assay.
"We often showed people that RPA could be successfully performed by stuffing the tube into an armpit, down a sock, or in similar locations that could provide the necessary heat."
Thus, for a low-resource setting, body heat RPA could reduce the equipment burden down to a hotplate for sample prep and some pipette tips, Crannell said, eliminating the need for a heater that can maintain a set temperature.
Niall Armes, CEO of TwistDx, said in an email that the company had alluded to, and occasionally demonstrated, body heat-activated techniques in the past. "We often showed people that RPA could be successfully performed by stuffing the tube into an armpit, down a sock, or in similar locations that could provide the necessary heat," he said.
RPA is "totally unique" among isothermal methods in that lower temperatures, and resulting slower reactions, do not lead to formation of primer dimers or other artifacts, Armes claimed.
Due to interplay between thermal conditions and time to result, there continue to be advantages to managing the thermal incubation with some degree of external control, he said. "Nonetheless these unique properties of RPA clearly distinguish [it] with special potential for very low-cost consumables and instrumentation, compatibility with fluidics systems for which precise thermal control is challenging, and even instrument-free solutions when that is the only option available."
This claim is also supported by other recent work which showed RPA could be done with innovative, low-cost heating.
For instance, in a study run by Seattle's PATH institute, the Fred Hutchinson Cancer Research Center, and TwistDx, RPA was incubated using an exothermic chemical reaction contained in a small lidded plastic container of the sort usually used in the food service industry. The study determined the $0.10 chemical heater, ultimately intended for use in an infant HIV diagnostic, could detect 10 copies of HIV-1 DNA in around 30 minutes. Importantly, that study also showed RPA could be run over a range of ambient temperatures typical of Sub-Saharan Africa, concluding that the chemical heater may only be required in cooler weather.
One of the authors of that study, David Boyle, a senior research scientist at PATH whose group published an isothermal assay for HIV-1 last year, said in an email that the Rice study was "really interesting in terms of an instrument-free methodology for RPA incubation, and in principle it could work in the field with 'patient power'." However, Boyle pointed out that sample prep is critical for RPA, and must be carefully addressed.
"PATH faced the same challenge when using the chemical heater and we have now developed chemistries that can incubate samples at 60° C to 90° C to liberate DNA/RNA prior to amplification, but this process adds time, reagents, and complexity regardless of the test method, making it more challenging in the field," Boyle noted. Since the purpose of point of care is to diagnose and begin treatment in a single provider-patient interaction, "more integrated stand-alone tools or batched testing have to be currently considered as more cost- and time-effective for NAAT testing outside of the traditional laboratory setting," he said. Patient willingness to perform self-incubation using the axilla method might also be a concern.
TwistDx was acquired by Alere in 2010 and its RPA chemistry plays a role in Alere projects such as the next-generation pandemic influenza test on the Alere i platform, Armes said. That project recently won a three-year contract worth up to $12.9 million from the US Department of Health and Human Services' Biomedical Advanced Research and Development Authority. The company also continues to accelerate programs to provide point-of-care diagnostic tests as part of its role within the Alere group including assays that will run on the Alere i and new platform systems under development, Armes noted.
TwistDx is also intensifying efforts to recruit customers and partners to exploit the RPA technology, particularly for the agriculture, veterinary, environmental testing, and forensics lab applied markets. "We encourage interested parties to contact TwistDx directly," Armes said.
Crannell, meanwhile, said the Rice group, which also recently published a quantitative RPA method, will soon publish an RPA-based Giardia assay, and is attempting to develop methods for multiplexing RPA.