Two-Stage Isothermal Enzymatic Amplification for Concurrent Multiplex Molecular Detection

Abstract

Abstract BACKGROUND The wide array of pathogens responsible for infectious diseases makes it difficult to identify causative pathogens with single-plex tests. Although multiplex PCR detects multiple targets, it is restricted to centralized laboratories, which delays test results or makes multiplexing unavailable, depriving healthcare providers of critical, real-time information. METHODS To address the need for point-of-care (POC) highly multiplexed tests, we propose the 2-stage, nested-like, rapid (<40 min) isothermal amplification assay, dubbed rapid amplification (RAMP). RAMP's first-stage uses outer loop-mediated isothermal amplification (LAMP) primers to amplify all targets with recombinase polymerase amplification (RPA). First-stage amplicons are aliquoted to second stage reactors, each specialized for a specific target, to undergo LAMP. The assay is implemented in a microfluidic chip. LAMP amplicons are detected in situ with colorimetric dye or with a fluorescent dye and a smartphone. RESULTS In experiments on a benchtop and in a microfluidic format, RAMP demonstrated high level of multiplexing (≥16); high sensitivity (i.e., 1 plaque-forming unit of Zika virus) and specificity (no false positives or negatives); speed (<40 min); ease of use; and ability to cope with minimally processed samples. CONCLUSIONS RAMP is a hybrid, 2-stage, rapid, and highly sensitive and specific assay with extensive multiplexing capabilities, combining the advantages of RPA and LAMP, while circumventing their respective shortcomings. RAMP can be used in the lab, but one of its distinct advantages is amenability to simple implementation in a microfluidic format for use at the POC, providing healthcare personnel with an inexpensive, highly sensitive tool to detect multiple pathogens in a single sample, on site.