Nucleic Acid Amplification of Individual Molecules in a Microfluidic Device

Dettloff, Roger; Yang, Esther; Rulison, Aaron J.; Chow, Andrea W.; Farinas, Javier

doi:10.1021/ac800339w

Cited by 19 publications

(21 citation statements)

References 16 publications

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“…Improvements have included faster amplification [20], [21], reduced sample and reagent consumption [22], [23], single-use chip materials [24] and increased integration of the PCR device with sample preparation and detection modules [25], [26], [27], [28], [29]. Only a few reports have demonstrated integrated chips with molecular diagnostic capabilities using complex clinical specimens [30], [31], [32].…”

Section: Introductionmentioning

confidence: 99%

Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens

et al. 2012

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A rapid, low cost, accurate point-of-care (POC) device to detect influenza virus is needed for effective treatment and control of both seasonal and pandemic strains. We developed a single-use microfluidic chip that integrates solid phase extraction (SPE) and molecular amplification via a reverse transcription polymerase chain reaction (RT-PCR) to amplify influenza virus type A RNA. We demonstrated the ability of the chip to amplify influenza A RNA in human nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens collected at two clinical sites from 2008–2010. The microfluidic test was dramatically more sensitive than two currently used rapid immunoassays and had high specificity that was essentially equivalent to the rapid assays and direct fluorescent antigen (DFA) testing. We report 96% (CI 89%,99%) sensitivity and 100% (CI 95%,100%) specificity compared to conventional (bench top) RT-PCR based on the testing of n = 146 specimens (positive predictive value = 100% (CI 94%,100%) and negative predictive value = 96% (CI 88%,98%) ). These results compare well with DFA performed on samples taken during the same time period (98% (CI 91%,100%) sensitivity and 96% (CI 86%,99%) specificity compared to our gold standard testing). Rapid immunoassay tests on samples taken during the enrollment period were less reliable (49% (CI 38%,61%) sensitivity and 98% (CI 98%,100%) specificity). The microfluidic test extracted and amplified influenza A RNA directly from clinical specimens with viral loads down to 10 3 copies/ml in 3 h or less. The new test represents a major improvement over viral culture in terms of turn around time, over rapid immunoassay tests in terms of sensitivity, and over bench top RT-PCR and DFA in terms of ease of use and portability.

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Section: Introductionmentioning

confidence: 99%

Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens

et al. 2012

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“…Application of Poisson distribution as a statistical tool for this purpose was suggested by Wang and Spadoro (47) for conventional PCR. In general, the analysis of positive and negative results in a cohort of samples was used by incidence of rare events such as frequency of alleles, mutations, levels of viruses, and clone frequencies in complex populations (1,6,11,22,34,35,41), and by several studies presenting new nucleic acid-based methods (5,9,12,14,19). Nevertheless, Poisson analysis was applied qualitatively, and the quantitative information was neglected.…”

Section: Discussionmentioning

confidence: 99%

A Novel Poisson Distribution-Based Approach for Testing Boundaries of Real-Time PCR Assays for Food Pathogen Quantification

Rossmanith

Wagner

2011

Journal of Food Protection

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The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in food pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.

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“…As proved also by clinical standards, PCR is an invaluable tool for nucleic acids analysis, especially in viral diseases and cancer diagnosis [56]. Analysis of heterogeneous nucleic acid mixtures is a source of important clinical information, especially in assessment of antiretroviral resistant mutations or early cancer detection [57]. Individually, genotypic investigation of minority viral populations might be a source of valuable information such as the tendency to develop drug resistance.…”

Section: Pcr On a Chipmentioning

confidence: 99%

“…The feasibility of rapid, single-molecule amplification of nucleic acids was established using a microfluidic system developed to enable rapid PCR analysis of individual DNA molecules with precise temperature control [57]. PCR was performed on heterogeneous samples containing synthetic CYP2D6.6 wild-type and mutant templates, on a quartz chip designed to enable adequate mixing by Brownian diffusion after each stage of reagent dispensing.…”

Section: Pcr On a Chipmentioning

confidence: 99%

Microfluidics in macro-biomolecules analysis: macro inside in a nano world

et al. 2010

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Use of microfluidic devices in the life sciences and medicine has created the possibility of performing investigations at the molecular level. Moreover, microfluidic devices are also part of the technological framework that has enabled a new type of scientific information to be revealed, i.e. that based on intensive screening of complete sets of gene and protein sequences. A deeper bioanalytical perspective may provide quantitative and qualitative tools, enabling study of various diseases and, eventually, may offer support for the development of accurate and reliable methods for clinical assessment. This would open the way to molecule-based diagnostics, i.e. establish accurate diagnosis and disease prognosis based on identification and/or quantification of biomacromolecules, for example proteins or nucleic acids. Finally, the development of disposable and portable devices for molecule-based diagnosis would provide the perfect translation of the science behind life-science research into practical applications dedicated to patients and health practitioners. This review provides an analytical perspective of the impact of microfluidics on the detection and characterization of bio-macromolecules involved in pathological processes. The main features of molecule-based diagnostics and the specific requirements for the diagnostic devices are discussed. Further, the techniques currently used for testing bio-macromolecules for potential diagnostic purposes are identified, emphasizing the newest developments. Subsequently, the challenges of this type of application and the status of commercially available devices are highlighted, and future trends are noted.

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Nucleic Acid Amplification of Individual Molecules in a Microfluidic Device

Cited by 19 publications

References 16 publications

Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens

Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens

A Novel Poisson Distribution-Based Approach for Testing Boundaries of Real-Time PCR Assays for Food Pathogen Quantification

Microfluidics in macro-biomolecules analysis: macro inside in a nano world

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