Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2014

A SYBR-GREEN assay for recombinase polymerase amplification screening, and development of a rapid assay for Murray Valley Encephalitis (#327)

Joanna Kristoffersen 1 , Jack Simpson 2 , Pavithra Vijayakumar 3 , Natalie Prow 4 , Joanne Macdonald 1 5
  1. Inflammation and Healing Research Cluster, University of the Sunshine Coast, Maroochydore, QLD, Australia
  2. Australian National University, Canberra, ACT, Australia
  3. Princeton University, Princeton, NJ, USA
  4. School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia
  5. Division of Experimental Therapeutics, Columbia University, New York, NY, USA

Recombinase polymerase amplification (RPA) is a relatively new isothermal amplification technique offering particular advantages for development of point-of-care assays. In particular, the single temperature of incubation (37-39 °C), the freeze-dried components, and the ability to link with lateral flow device detection are all excellent features for use in low resource settings. Unlike traditional PCR, where heating and cooling enables insertion of primers into a double stranded template, in RPA the primer is enzymatically inserted. Traditional PCR primers make inefficient substrates for this process, rather, primers need to be tailored to suit recombinase insertion. Some traits have been identified that enable semi-efficient primer and probe design, such as increasing the length of the primer and avoiding long strings of guanines (Gs). However, these rules are not sufficiently comprehensive to enable optimal first-time designs. Thus, the current recommendation is to create at least 5 variations of each primer and probe, and iteratively test combinations, in a time-consuming and expensive process.

Here we have applied a SYBR-GREEN I assay to monitor the efficiency of RPA amplification in real-time. The process was applied to the development of an RPA assay for detection of the Murray Valley encephalitis virus (MVEV) NS5 gene. This virus is endemic in northern Australia, and incursions to southern states results in outbreaks, with associated human morbidity and mortality. A rapid field-test for MVEV could aid monitoring of disease and improve decision-making during patient treatment. By implementing the SYBR-GREEN screening of primers and probes, combined with gel electrophoresis, we were easily able to identify the optimal combination of primers that allowed specific amplification of MVEV RNA. Our final assay efficiently amplified MVEV RNA template in 15 minutes, followed by detection in 5 minutes using a lateral flow device. Our SYBR-GREEN primer screening assay enables fast, efficient, and cost-effective primer screening for RPA development.