In a study published in PLOS Biology, the researchers initially investigated the ability of certain chemicals to capture anionic DNA and RNA by spotting them on a piece of cellulose-based paper. While none of the chemical treatments produced reproducible amplification, the team found that the nucleic acids almost instantly bound to the untreated paper.
In addition, the cellulose-based paper retained the genetic material during a short washing step to remove contaminants, and they then eluted the material directly into an amplification reaction chamber.
"We used the adsorbing solvency of the paper to suck up a certain amount of the genetic sample, as determined by the size of the nucleic acid binding site," Michael Mason, a University of Queensland senior postdoctoral research fellow and co-first author on the paper, said in an interview.
Mason's team adapted the cellulose filter to develop an equipment-free nucleic acid extraction process that uses a dipstick made from the paper, with a small 8-mm2 DNA-binding surface and a long water-repellent handle made by filling the filter paper with paraplast wax.
The process involves homogenizing tissue in a tube containing appropriate lysis buffer and ball bearings to help break down the tissue, then using the dipstick to capture nucleic acids by dipping it into the lysate three times. The dipstick is then dipped up and down in a wash solution three times. Finally, the bound nucleic acids are eluted from the cellulose by putting the dipstick directly into the amplification mix three times.
While researchers have used other types of membranes to induce rapid nucleic acid extraction, including aluminum oxide and silica-based filters, the methods require relatively complex fabrication, multiple pipetting steps, or electrical equipment to help purify the nucleic acids.
To validate the new nucleic acid purification method, Mason's team matched the test against AMPure, a popular commercial rapid paramagnetic bead DNA extraction method sold by Beckman Coulter. They saw that the cellulose method purified amplifiable DNA significantly faster, in under 30 seconds compared to 14.5 minutes for AMPure purification.
While techniques like AMPure only take 10 minutes and do not require electrical equipment, Mason's team believes the tools required are too costly for external applications such as field-based point-of-need assays.
"Importantly, the method achieves this speed and simplicity without the need for any pipetting, [and is] significantly cheaper, with consumables costing four times less than those required by the AMPure system, and does not require the initial investment of $685-$876 for the specialized magnet plate," the authors stated in the paper.
According to the study, AMPure's paramagnetic beads cost $0.65 per sample, whereas the team's cellulose dipstick purification method costs $0.15 per sample, including plasticware and reagents. Mason explained that if the ball bearings used to homogenize the tissue are washed and reused, the cost for the cellulose dipstick purification could be further reduced to $0.06 per sample.
Mason and his team have filed a patent for the cellulose disc extraction method through UniQuest, a commercialization company for the University of Queensland. Mason said that UniQuest is currently in discussions with firms interested in commercializing the technology, but did not disclose them at this time.
"We haven't found [any] limitations, but we are looking into new uses for this technology, and we might find that we do need to increase the amount of DNA that binds onto the dipstick," Mason said.
Mason envisions the cellulose-based technology to be used in a wide range of situations, including clinical care and high school science classes. He also plans to use the technology to detect food pathogens, and his team is currently working with food-based molecular diagnostic projects in Papua New Guinea.
"You can have situations in the near future, like in hospitals, if a doctor suspects you have some sort of blood pathogen, he could actually take a drop of blood and perform the test right next to the bedside," Mason said.
In addition, Mason built an electronic amplification device that cost about A$30 ($23) using parts purchased on Ebay. He explained that this device "takes the tube out of the dipstick, performs the reactions, and returns the results quickly." Mason said that "doctors would know within an hour what's wrong with the patient."