to BioTechniques free email alert service to receive content updates.
Evaluation of Isothermal Recombinase Polymerase Amplification Incubation Temperature Tolerance
 
BioTechniques, Vol. 61, No. 6, December 2016, p. 328
Full Text (PDF)

Overview

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.

Summary

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).


Figure 1. 


TwistAmp® users have previously reported similar flexibility in RPA temperature requirements with HIV (1, 2) and Schistosoma japonicum (3) targets.

The whole range of TwistAmp® products can be purchased at www.twistdx.co.uk/products or over the phone at +44 (0)1223 496700.

References