Interfacial Effect-Based Quantification of Droplet Isothermal Nucleic Acid Amplification for Bacterial Infection

Sci Rep. 2019 Jul 3;9(1):9629. doi: 10.1038/s41598-019-46028-8.

Abstract

Bacterial infection is a widespread problem in humans that can potentially lead to hospitalization and morbidity. The largest obstacle for physicians/clinicians is the time delay in accurately identifying infectious bacteria, especially their sub-species, in order to adequately treat and diagnose such infected patients. Loop-mediated amplification (LAMP) is a nucleic acid amplification method that has been widely used in diagnostic applications due to its simplicity of constant temperature, use of up to 4 to 6 primers (rendering it highly specific), and capability of amplifying low copies of target sequences. Use of interfacial effect-based monitoring is expected to dramatically shorten the time-to-results of nucleic acid amplification techniques. In this work, we developed a LAMP-based point-of-care platform for detection of bacterial infection, utilizing smartphone measurement of contact angle from oil-immersed droplet LAMP reactions. Whole bacteria (Escherichia coli O157:H7) were assayed in buffer as well as 5% diluted human whole blood. Monitoring of droplet LAMP reactions was demonstrated in a three-compartment, isothermal proportional-integrated-derived (PID)-controlled chip. Smartphone-captured images of droplet LAMP reactions, and their contact angles, were evaluated. Contact angle decreased substantially upon target amplification in both buffer and whole blood samples. In comparison, no-target control (NTC) droplets remained stable throughout the 30 min isothermal reactions. These results were explained by the pre-adsorption of plasma proteins to an oil-water interface (lowering contact angle), followed by time-dependent amplicon formation and their preferential adsorption to the plasma protein-occupied oil-water interface. Time-to-results was as fast as 5 min, allowing physicians to quickly make their decision for infected patients. The developed assay demonstrated quantification of bacteria concentration, with a limit-of-detection at 102 CFU/μL for buffer samples, and binary target or no-target identification with a limit-of-detection at 10 CFU/μL for 5% diluted whole blood samples.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacteria / genetics*
  • Bacterial Infections / diagnosis*
  • Bacterial Infections / microbiology*
  • Escherichia coli O157 / genetics
  • Humans
  • Nucleic Acid Amplification Techniques* / instrumentation
  • Nucleic Acid Amplification Techniques* / methods
  • Nucleic Acid Amplification Techniques* / standards
  • Point-of-Care Testing
  • Sensitivity and Specificity