Glass-composite prototyping for flow PCR with in situ DNA analysis

Pjescic, Ilija; Tranter, Collin; Hindmarsh, Patrick L.; Crews, Niel

doi:10.1007/s10544-009-9389-2

Cited by 41 publications

(8 citation statements)

References 18 publications

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“…The combination of two single reactions into one microfluidic reactor represents high system integration in comparison to other publications (Sun et al 2008;Hartung et al 2009). In contrast to prototype devices (Pjescic et al 2010;Ramalingam et al 2010), the established fabrication method is capable of large-scale production, which is crucial when used as disposable device. Furthermore, this potential to dispose the chip after the analysis is crucial, when the chip should be used as an IVD test.…”

Section: Discussionmentioning

confidence: 99%

Disposable microfluidic chip for rapid pathogen identification with DNA microarrays

et al. 2011

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This work presents the combination and acceleration of PCR and fluorescent labelling within a disposable microfluidic chip. The utilised geometry consists of a spiral meander with 40 turns, representing a cyclic-flow PCR system. The used reaction chemistry includes Cy3-conjugated primers leading to a one-step process accelerated by cyclic-flow PCR. DNA of three different bacterial samples (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) was processed and successfully amplified and labelled with detection limits down to 10 2 cells per reaction. The specificity of species identification was comparable to the approach of separate PCR and labelling. The overall processing time was decreased from 6 to 1.5 h. We showed that a disposable polycarbonate chip, fabricated by injection moulding is suitable for the significant acceleration of DNA microarray assays. The reaction output led to high-sensitivity bacterial identification in a short time, which is crucial for an early and targeted therapy against infectious diseases.

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Section: Discussionmentioning

confidence: 99%

Disposable microfluidic chip for rapid pathogen identification with DNA microarrays

et al. 2011

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“…In contrast to the amplification of phage DNA (Pjescic et al 2010;Dorfman et al 2005) or abundant DNA templates (Obeid et al 2003), this approach is applicable to the detection of minute amounts of pathogenic bacteria. Furthermore a reduced sensitivity compared to conventional thermocyclers (Sauer-Budge et al 2009;Park et al 2003) was not observed using our microfluidic device.…”

Section: Limit Of Detectionmentioning

confidence: 99%

“…Most literature demonstrate a proof of principle without concentrating on the limits and drawbacks important for a particular application. The major constraints are the restriction to phage DNA (Pjescic et al 2010;Dorfman et al 2005), limited amplicon lengths (Mahalanabis et al 2010;Zhang and Xing 2010;Li et al 2009), lower sensitivity compared to conventional thermocyclers (Sauer-Budge et al 2009;Park et al 2003) and the restriction to abundant DNA templates (Obeid et al 2003). In aspect of species identification by DNA microarrays, long targets are especially important for the accurate discrimination between different bacteria (Wiesinger-Mayr et al 2007;Yu et al 2004).…”

Section: Introductionmentioning

confidence: 99%

Long target droplet polymerase chain reaction with a microfluidic device for high-throughput detection of pathogenic bacteria at clinical sensitivity

Peham

Grienauer

Steiner

et al. 2011

Biomed Microdevices

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In this article we present a long target droplet polymerase chain reaction (PCR) microsystem for the amplification of the 16S ribosomal RNA gene. It is used for detecting Gram-positive and Gram-negative pathogens at high-throughput and is optimised for downstream species identification. The miniaturised device consists of three heating plates for denaturation, annealing and extension arranged to form a triangular prism. Around this prism a fluoropolymeric tubing is coiled, which represents the reactor. The source DNA was thermally isolated from bacterial cells without any purification, which proved the robustness of the system. Long target sequences up to 1.3 kbp from Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa have successfully been amplified, which is crucial for the successive species classification with DNA microarrays at high accuracy. In addition to the kilobase amplicon, detection limits down to DNA concentrations equivalent to 10(2) bacterial cells per reaction were achieved, which qualifies the microfluidic device for clinical applications. PCR efficiency could be increased up to 2-fold and the total processing time was accelerated 3-fold in comparison to a conventional thermocycler. Besides this speed-up, the device operates in continuous mode with consecutive droplets, offering a maximal throughput of 80 samples per hour in a single reactor. Therefore we have overcome the trade-off between target length, sensitivity and throughput, existing in present literature. This qualifies the device for the application in species identification by PCR and microarray technology with high sample numbers. Moreover early diagnosis of infectious diseases can be implemented, allowing immediate species specific antibiotic treatment. Finally this can improve patient convalescence significantly.

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“…Typically, an additional postprocessing step that takes about 30 min is required by existing NAT devices to obtain melt curves. Previously, we have reported the acquisition of amplification and melt curves using fluorescence images while thermocycling the sample (Crews, Wittwer, & Gale, 2008; Crews, Wittwer, Montgomery, Pryor, & Gale, 2009; Pjescic & Crews, 2012; Pješčić, Tranter, Hindmarsh, & Crews, 2010). This system was based on continuous flow in a thermal gradient, eliminating the need for stringent temperature control.…”

Section: Introductionmentioning

confidence: 99%

A versatile oscillating‐flow microfluidic PCR system utilizing a thermal gradient for nucleic acid analysis

Kopparthy

Crews

2020

Biotech & Bioengineering

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We report the development of a versatile system based on the oscillating‐flow methodology in a thermal gradient system for nucleic acid analysis. Analysis of DNA and RNA samples were performed in the device, without additional temperature control and complexity. The technique reported in this study eliminates the need for predetermined fluidic channels for thermocycles, and complexity involved with additional incubation steps required for RNA amplification. A microfluidic device was fabricated using rapid prototyping by simply sandwiching dual side adhesive Kapton tape and a polydimethylsiloxane spacer between glass microscope slides. Amplification of the 181‐bp segment of a viral phage DNA (ΦX174) and B2M gene in human RNA samples was demonstrated using the system. The developed system enables simultaneous acquisition of amplification and melt curves, eliminating the need for postprocessing. A direct comparison between the oscillating‐flow system and a commercial real‐time polymerase chain reaction (PCR) instrument showed complete agreement in PCR data and improved sample‐to‐result time by eliminating an additional 30 min melt curve step required in commercial PCR systems.

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Glass-composite prototyping for flow PCR with in situ DNA analysis

Cited by 41 publications

References 18 publications

Disposable microfluidic chip for rapid pathogen identification with DNA microarrays

Disposable microfluidic chip for rapid pathogen identification with DNA microarrays

Long target droplet polymerase chain reaction with a microfluidic device for high-throughput detection of pathogenic bacteria at clinical sensitivity

A versatile oscillating‐flow microfluidic PCR system utilizing a thermal gradient for nucleic acid analysis

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