An alternative to PCR that can provide mobile, on-site testing by first responders has been developed by TwistDx.
Based on recombinase polymerase amplification (RPA) isothermal nucleic acid amplification and detection technology, it can detect pathogens, transgenes, and genetic markers within five to 10 minutes, making diagnosis possible within about 30 minutes of sample collection, according to the company.
Compared to PCR, RPA “operates without a need for thermocycling and at a wide temperature range,” explained Helen Kent, operations manager.
“RPA may achieve additional speed by the fact that reiterative primer-elongation events do not require completion of synthesis from the opposing side, as in PCR. In this way, amplification combines both a reiterative linear round of replication and strand displacement with geometric-phase aspects driven by reconversion of single-strands to duplexes.”
Additionally, RPA synthesizes defined points in sample DNA, so amplification occurs only if the target region is present, she explained. “RPA can amplify to detectable levels, at optimal temperature, in only six minutes from single-target levels.
“The entire reaction system is stable as a dried formulation and can be transported safely without refrigeration,” according to TwistDx. Other benefits include the ability to function on crude samples and low, constant-temperature operation.
For example, the optimal operation temperature is 37ºC, but the process will work—albeit more slowly—at a typical ambient temperature of 25ºC. Therefore, it may be possible to develop a simple, inexpensive disposable test system for use in cold environments or field conditions, Kent said.
Sensitivity is such that RPA can detect single copies of DNA and 10 copies of RNA. That is sufficient, TwistDx says, to operate at the single molecule level in the presence of hundreds of nanograms of unrelated complex genomic DNA, like that from plants and mammals. Several different targets may be detected with a single test because multiple detection primers can be combined in one tube.
DVDs Meet Biosensing
At Philips Research, Reinhold Wimberger-Friedl and his team have developed a compact fluorescence reader based on confocal scanning with submicron resolution and single fluorophore sensitivity, which could be taken into the field or used in small labs.
“Since the main optical components are derived from optical storage technology, the instrument can be as compact and low cost as a portable DVD player,” he said. And, it is robust and easy to use, he added.
“As a demonstration, we have developed a simple sandwich assay for C-reactive protein with printed spots on a plastic substrate and fluorescently labeled antibodies. We were able to detect 100 fM without further assay optimization,” Dr. Wimberger-Friedl said. The device is well-suited for real-time hybridization studies because it “allows detection of bound targets at the surface with low interference from background noise originating from the solution above the surface.”
Essentially, the system is an x-y scanner with moving optics and a static object or substrate. It operates in reflective mode through the substrate. “In combination with confocal detection, background signals are suppressed, which allows real-time hybridization measurements. The auto-focus capability makes the system robust,” Dr. Wimberger-Friedl reported.
The device minimizes background fluorescence and provides a low-cost instrument for the laboratory and for less experienced users, according to Dr. Wimberger-Friedl. The technology is based upon Philips’ pioneering DVD work, adapted to biosensing.
“We set ourselves the goal of achieving single binding event resolution so that we could do digital biosensing much like the digital optical recording. This would give us the highest sensitivity and dynamic range,” Dr. Wimberger-Friedl said.