By Ben Butkus
Taiwanese researchers have developed a method of performing PCR using natural convection and a single isothermal heater, and have used the technique to amplify DNA from three different viral genomes with sensitivity comparable to that of existing PCR methods.
According to the scientists, the new method may be able to serve as the basis for extremely inexpensive and easy-to-use molecular diagnostic tests in developing nations since it eliminates the need for expensive and delicate thermal cyclers.
In an e-mail to PCR Insider, Pei-Jer Chen, one of several research team members from National Taiwan University, said that he and his colleagues came up with the method after having tried for years to develop suitable microfluidic chips for PCR to no avail.
For example, the group had previously developed a microcirculating PCR chip that used a suction-type membrane for fluidic transport. And indeed, many academic research labs and companies have developed or are developing microfluidic chips for PCR with the intent of employing them as part of molecular testing devices in developing nations.
However, devices such as these necessitate that reaction buffers run through tiny channels, which "impedes a smooth and rapid flow rate," Chen said. The Taiwanese researchers also found that microfluidic approaches required three temperature control zones to achieve thermocycling, thereby complicating device geometry.
"In addition, we need to maintain heating below and above the chip to keep the temperature in the buffer homogenous," Chen said. As such, microfluidic devices "can be painstakingly developed as the prototype" for POC testing platforms, "but are not readily useful," he added.
Reporting in the January edition of BioTechniques, the researchers from National Taiwan University in Taipei describe how they performed their method, which they dubbed capillary convective PCR, using only a standard capillary tube mounted on a dry heater fixed at 95°C.
Specifically, the continuously heated capillary base drives the lowest part of the sample to rise by convection while simultaneously denaturing the DNA template, the researchers explained. As the sample rises, its temperature falls due to cooling from surrounding air and, when it reaches a "cool temperature zone" near the top of the tube, it undergoes annealing and extension.
Finally, the DNA template sinks to the bottom of the tube to be heated again, thereby allowing PCR cycling via natural convection.
The scientists also demonstrated the importance of optimizing primer and amplicon design to successfully amplify DNA using the ccPCR method. Specifically, they showed that altering the melting temperature of the primers relative to the lowest temperature in the tube affected amplification efficiency; and that adjusting the amplicon denaturation temperature relative to the highest temperature in the tube affects maximum amplicon size.
"As this convective PCR only allows a constant period for DNA denaturation, renaturation, and DNA synthesis, the melting temperature for primers must be higher than the temperature at the top of reagent" by about 8°C, and should lie in a range of 76 to 78°C, Chen told PCR Insider. "Other than this point, the criteria for designing the primers are similar to conventional PCR," he added.
Using these criteria, the researchers were able to amplify several different amplicons of less than or equal to about 500 base pairs from viruses such as hepatitis C, human papillomavirus, and HIV, with a maximum sensitivity of about 30 copies per reaction, in about 30 minutes. Chen noted that because the DNA synthesis period is short in ccPCR, the group recommends that amplicons not exceed 500 to 600 base pairs.
He also said that the group has used the technique to reliably detect target DNA at 10 copies per reaction, "but variably at three to five copies," although that data has not yet been published. These numbers are comparable to most PCR methods on the market or in development, except for methods specializing in detecting extremely low or even single copy numbers, such as digital PCR.
The researchers also noted in their report that other groups have attempted to conduct PCR using natural convection, but that most of these devices have still required two or more independent temperature controllers and complicated designs requiring specific tube shapes or additional chambers to circulate fluids completely.
In contrast, the National Taiwan University researchers demonstrated that their method could be performed using simple heating systems even of the kind not usually found in laboratories — for instance, a hot-water bath or the heating plate of a portable scent-based mosquito repellent device. Indeed, "ccPCR can work on many home appliances, and really does not need a laboratory water bath," Chen said.
"With its advantages of low cost and ease of operation, ccPCR would be well-suited for less-developed countries hoping to improve surveillance systems for disease outbreaks," the researchers noted in their report. "We also hope that ccPCR can make nucleic acid testing more accessible beyond laboratories by facilitating point-of-care applications."
One major hurdle to this becoming a reality is the possible effect of ambient temperature on ccPCR, since the method uses ambient temperature as the cooling mechanism, the researchers noted. In their tests, the technique was effective both at 4°C and at 33°C in an incubator, but failed when the ambient temperature was greater than 38°C — a real concern in many developing parts of the world where a ccPCR-based molecular testing platform might come in handy.
"To solve this, either we need to find a slightly cool spot or time to run the reaction, or use some heat dissipation device," Chen said.
The researchers initially applied for a patent on their technology through National Taiwan University, but it was rejected, Chen said. The group then enlisted the help of an undisclosed private entity, which has applied for PCT patents covering the technology, he added.
Chen said that the group does not yet have a formal commercialization plan in place for ccPCR, but hopes that it can find industry collaborators. He also said that an undisclosed Taiwanese company is attempting to develop the technology for use in veterinary diagnostics.
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