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

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/?L for buffer samples, and binary target or no-target identification with a limit-of-detection at 10?CFU/?L for 5% diluted whole blood samples.

SUBMITTER: Ulep TH

PROVIDER: S-EPMC6610113 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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

Ulep Tiffany-Heather TH Day Alexander S AS Sosnowski Katelyn K Shumaker Alexa A Yoon Jeong-Yeol JY

Scientific reports 20190703 1

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 ...[more]

PMID: 31270374

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Project description:Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70-120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99.3%, and a PPV of 84% and NPV of 98% as compared to clinical microbial detection methods. Results indicate the LAMP-based In-Dx panel allows rapid and precise diagnosis of clinical infections by targeted pathogens across multiple culture types for point-of-care utilization.

Dataset Information

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

Publications

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

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