Amplicon Competition Enables End‐Point Quantitation of Nucleic Acids Following Isothermal Amplification

Jiang, Yu; Stacy, Apollo; Whiteley, Marvin; Ellington, Andrew D.; Bhadra, Sanchita

doi:10.1002/cbic.201700317

Cited by 16 publications

(12 citation statements)

References 39 publications

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“…Recently, we engineered these molecular innovations to function fluently in one-pot LAMP-OSD reactions that can directly amplify a few tens to hundreds of copies of DNA and RNA analytes from minimally processed specimens and produce sequence-specific fluorescence signals that are easily observable by the human eye or (more importantly) by unmodified cellphone cameras [ 42 ]. The fluorescence endpoints that are produced can be used for yes/no determinations of the presence of an analyte, and also estimation of analyte copies on an order of magnitude scale [ 42 , 43 ].…”

Section: Introductionmentioning

confidence: 99%

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone

et al. 2018

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Manipulation of natural mosquito populations using the endosymbiotic bacteria Wolbachia is being investigated as a novel strategy to reduce the burden of mosquito-borne viruses. To evaluate the efficacy of these interventions, it will be critical to determine Wolbachia infection frequencies in Aedes aegypti mosquito populations. However, current diagnostic tools are not well-suited to fit this need. Morphological methods cannot identify Wolbachia, immunoassays often suffer from low sensitivity and poor throughput, while PCR and spectroscopy require complex instruments and technical expertise, which restrict their use to centralized laboratories. To address this unmet need, we have used loop-mediated isothermal amplification (LAMP) and oligonucleotide strand displacement (OSD) probes to create a one-pot sample-to-answer nucleic acid diagnostic platform for vector and symbiont surveillance. LAMP-OSD assays can directly amplify target nucleic acids from macerated mosquitoes without requiring nucleic acid purification and yield specific single endpoint yes/no fluorescence signals that are observable to eye or by cellphone camera. We demonstrate cellphone-imaged LAMP-OSD tests for two targets, the Aedes aegypti cytochrome oxidase I (coi) gene and the Wolbachia surface protein (wsp) gene, and show a limit of detection of 4 and 40 target DNA copies, respectively. In a blinded test of 90 field-caught mosquitoes, the coi LAMP-OSD assay demonstrated 98% specificity and 97% sensitivity in identifying Ae. aegypti mosquitoes even after 3 weeks of storage without desiccant at 37°C. Similarly, the wsp LAMP-OSD assay readily identified the wAlbB Wolbachia strain in field-collected Aedes albopictus mosquitoes without generating any false positive signals. Modest technology requirements, minimal execution steps, simple binary readout, and robust accuracy make the LAMP-OSD-to-cellphone assay platform well suited for field vector surveillance in austere or resource-limited conditions.

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Section: Introductionmentioning

confidence: 99%

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone

et al. 2018

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“…One criticism that might be raised is the inability of HF183 LAMP-OSD to provide a quantitative determination of the level of contamination. To address this concern we have recently developed a robust, field-usable technology for semi-quantitative LAMP that enables reliable measurement of initial target copies on an order of magnitude scale via a simple, one-time determination of the presence or absence of visible OSD fluorescence at reaction endpoint (Jiang et al 2017). OSD fluorescence amplitude is rendered a function of initial target copies by diverting replicative resources from these true targets to defined numbers of competing ‘false’ targets that display the same primer binding sites and amplification kinetics but lack OSD complementarity.…”

Section: Discussionmentioning

confidence: 99%

Portable platform for rapid in-field identification of human fecal pollution in water

et al. 2018

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Human fecal contamination of water is a public health risk. However, inadequate testing solutions frustrate timely, actionable monitoring. Bacterial culture-based methods are simple but typically cannot distinguish fecal host source. PCR assays can identify host sources but require expertise and infrastructure. To bridge this gap we have developed a field-ready nucleic acid diagnostic platform and rapid sample preparation methods that enable on-site identification of human fecal contamination within 80 min of sampling. Our platform relies on loop-mediated isothermal amplification (LAMP) of human-associated Bacteroides HF183 genetic markers from crude samples. Oligonucleotide strand exchange (OSD) probes reduce false positives by sequence specifically transducing LAMP amplicons into visible fluorescence that can be photographed by unmodified smartphones. Our assay can detect as few as 17 copies/ml of human-associated HF183 targets in sewage-contaminated water without cross-reaction with canine or feline feces. It performs robustly with a variety of environmental water sources and with raw sewage. We have also developed lyophilized assays and inexpensive 3D-printed devices to minimize cost and facilitate field application.

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“…Other systems utilized fluorescently labeled oligonucleotide strand displacement probes that are incorporated into LAMP amplicons ( Bhadra et al, 2015 , 2018 ; Jiang et al, 2017 ; Priye et al, 2017 ). In an RT-LAMP assay for the multiplexed detection of Zika, dengue, and chikungunya viruses in urine samples, probes for each virus were labeled with a different dye to produce a color-coded fluorescent readout ( Yaren et al, 2017 ).…”

Section: Loop-mediated Isothermal Amplificationmentioning

confidence: 99%

Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics

Pumford

Spaczai

et al. 2020

Biosensors and Bioelectronics

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Early disease detection through point-of-care (POC) testing is vital for quickly treating patients and preventing the spread of harmful pathogens. Disease diagnosis is generally accomplished using quantitative polymerase chain reaction (qPCR) to amplify nucleic acids in patient samples, permitting detection even at low target concentrations. However, qPCR requires expensive equipment, trained personnel, and significant time. These resources are not available in POC settings, driving researchers to instead utilize isothermal amplification, conducted at a single temperature, as an alternative. Common isothermal amplification methods include loop-mediated isothermal amplification, recombinase polymerase amplification, rolling circle amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. There has been a growing interest in combining such amplification methods with POC detection methods to enable the development of diagnostic tests that are well suited for resource-limited settings as well as developed countries performing mass screenings. Exciting developments have been made in the integration of these two research areas due to the significant impact that such approaches can have on healthcare. This review will primarily focus on advances made by North American research groups between 2015 and June 2020, and will emphasize integrated approaches that reduce user steps, reliance on expensive equipment, and the system's time-to-result.

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Amplicon Competition Enables End‐Point Quantitation of Nucleic Acids Following Isothermal Amplification

Cited by 16 publications

References 39 publications

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone

Portable platform for rapid in-field identification of human fecal pollution in water

Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics

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