Researchers in Spain are using Recombinase Polymerase Amplification (RPA) as the foundation for a highly sensitive and specific solid-phase optical assay that can detect the potential biowarfare agent, Y. pestis, in less than an hour.
The enzyme-linked oligonucleotide assay (ELONA) approach developed by Ioanis Katakis and Ciara K. O’Sullivan, at the Universitat Rovira i Virgili’s Interfibio Research Group, and the ICREA (Catalan Institution for Research and Advanced Studies), uses conventional PCR primers to amplify both single- and double-stranded Y. pestis DNA. Their work provides proof of concept for applying RPA in a heterogeneous format, with one primer immobilized onto a solid surface. The researchers aim to further develop the technology into an integrated, portable lateral flow-type test device for the rapid amplification and detection of Y. pestis in resource limited and field settings for biodefence.
Y. pestis causes bubonic and pneumonic plague, and is lethal in up to 60% of cases that don’t receive swift treatment. The organism is considered by the US Centers for Disease Control and Prevention to represent a potential biowarfare agent. Although prompt diagnosis of Y. pestis infection facilitates expedited treatment, the current gold standard for detecting plague is time consuming, and involves bacterial isolation.
The quantitative detection method reported by Ioanis Katakis and colleagues exploits RPA to enable amplification of Y.pestis DNA in 30 minutes, and visual detection within the hour. The heterogeneous assay format uses a thiolated forward primer immobilized on the surface of maleimide-activated microtitre plates. The solid-phase RPA reaction is carried out in the wells at 37°C, in the presence of a solution-phase biotinylated reverse primer and either single-stranded or double-stranded DNA template. A streptavidin-HRP labelled probe detects the amplified target.
The researchers first tested their primers using conventional PCR to amplify synthetic and genomic Y. pestis DNA. They then applied the same primers to solution-phase RPA technology, again to amplify synthetic and genomic template. The PCR primers were subsequently used for the heterogeneous RPA method, which was carried out first with synthetic template during the development phase, and subsequently using genomic DNA from samples as proof of concept. Negative control assays included either incorporating no template DNA in the RPA mix, or using a non-specific DNA sequence, or a non-specific forward primer.
Results confirmed that the solid-phase RPA method was as sensitive and specific as PCR. ‘It was a real benefit to be able to use conventional PCR primers, rather than having to design longer, more expensive primers,’ Dr. Katakis notes. ‘Shorter primers also equate to simpler assay development. In fact, we have found that short PCR primers also work very well with RPA in other molecular diagnostic projects that we are involved with, including HLA typing.’
Drs Katakis and O’Sullivan aim to incorporate the heterogeneous RPA method into a simple lateral flow-type device for detecting Y. pestis. ‘The technology has many benefits compared with PCR-based assays and also in comparison with solution-phase RPA,’ Dr Katakis notes. ‘Not only is RPA isothermal, which means that we don’t need an intelligent system for temperature cycling, but the isothermal temperature is low. The heterogeneous RPA method also requires fewer wash steps than solution-phase approaches, and only uses two primers, which is in contrast with other isothermal amplification methods that require four primers and more complicated reagent handling. The less complicated the technique, the lower the potential for things to go wrong. Troubleshooting becomes simpler, and, ultimately, time to market is shorter. From my perspective RPA isn’t just a personal preference, it is technologically the best amplification method to use.’ The team used TwistAmp® Basic for their assay.