Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification

Abstract

An emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 103 and 106 CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 106 CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.

Progression of LAMP as a Result of the COVID-19 Pandemic: Is PCR Finally Rivaled?

“…Various methods, such as the addition of DMSO (dimethyl sulfoxide) [ 33 , 34 , 35 ] to the reaction mix and the use of OSD (oligonucleotide strand displacement) probes [ 24 , 25 , 26 , 27 , 28 ], have been proven to improve LAMP accuracy. In particular, compartmentalization of the reaction through microfluidics [ 29 , 30 , 31 ] and emulsion [ 32 , 122 , 123 ] techniques have shown substantial success in improving accuracy by limiting the effects of contaminants. This has been applied to PCR reactions as well, most popularly through digital-droplet PCR (ddPCR), in which reactions are partitioned (using oil) into thousands of “droplets,” eliminating interference from reaction-inhibiting particles [ 124 ].…”

“…( A ) Compartmentalization of emulsion LAMP reactions. Reprinted with permission from [ 32 ]. Copyright 2021, Day et al, under Creative Commons 4.0 Attribution License.…”

“…For example, many research groups have utilized oligonucleotide-strand-displacement (OSD) probes, fluorophore-labeled nucleic-acid strands that are sequence-specific, rather than relying on non-specific-reaction byproducts [ 24 , 25 , 26 , 27 , 28 ]. Both microfluidics [ 29 , 30 , 31 ] and emulsion [ 32 ] reactions have also been applied to LAMP diagnostics to compartmentalize the reactions, once again improving accuracy. Adding dimethyl sulfoxide (DMSO) to the reaction has also been proven to increase LAMP’s analytical specificity and sensitivity [ 33 ] and clinical specificity and sensitivity [ 34 , 35 ].…”