Abstract

A New Two-Stage Isothermal Enzymatic Amplification for Multiplex Molecular Detection at the Point of Care

Haim Bau, Professor, University Of Pennsylvania

The wide array of pathogens responsible for infectious diseases and co-infections makes it difficult to identify causative pathogens with single-plex tests. Although multiplexed PCR detects multiple targets, it is restricted to centralized laboratories, which either delays test results or makes multiplexing unavailable, depriving healthcare providers of critical, timely information. To address the need for a point-of-care multiplexed test, we developed a new two-stage, nested-like, rapid isothermal-isothermal amplification assay, dubbed RAMP. RAMP’s first stage uses outer LAMP primers to amplify all targets with Recombinase Polymerase Amplification (RPA). First stage amplicons are then distributed to second stage reactors, each specialized for a specific target, to undergo Loop-Mediated Isothermal Amplification (LAMP). RAMP combines the advantages of RPA and LAMP, while overcoming their respective shortcomings. We describe: RAMP’s performance as a highly sensitive benchtop assay; its ability to cope with minimally-processed samples; and its simple implementation in microfluidic format, wherein first-stage products are automatically distributed to second stage reactors, and test results are read either by eye or with a Smartphone. We demonstrate a sixteen-plex RAMP (more is possible), including the detection of 1 PFU of Zika virus in urine and discrimination between African and Asian strains of the virus.