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.