Isothermal DNA/RNA amplification technology provides flexibility of use, particularly in the field. TwistDx’s proprietary Recombinase Polymerase Amplification (RPA) biochemistry enables such field use. Firstly, it permits transport of reagents (freeze-dried enzymatic pellets) at room temperature to point of testing, even in remote areas. Secondly, it allows amplification and detection of DNA/RNA using low cost, portable equipment. RPA works at an optimal temperature range of 39-42oC, typically in 3-15 minutes. Low temperature amplification reduces hardware requirements. The tolerance to further reduced temperatures was explored.Materials and Methods
TwistAmp® Basic kit (50μl) reactions were prepared as follows:
Each freeze-dried pellet was rehydrated with 29.5μl rehydration buffer, 7μl primer mix (Basic kit positive control), 8.9μl dH20, 3.6μl magnesium acetate (280mM), 1μl of positive control template (250 copies/ul) or 1μl dH20 for no template control (ntc).
Reactions were incubated for an hour at a temperature of 25, 30, 35, 40, or 45oC in a heat block, reactions were briefly agitated after 4 minutes incubation.
Post amplification, reactions were cleaned with a standard PCR clean-up kit and run on a 2% agarose gel.
All reactions amplified a correctly sized product, at all the incubation temperatures tested. This flexible working temperature provides further evidence of RPA suitability for field work. A lower temperature requirement means lower power requirements - key for field applications. Furthermore RPA technology can be applied simply in a lateral flow format (TwistAmp® nfo).
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