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Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment.

Keller M, Naue J, Zengerle R, von Stetten F, Schmidt U - PLoS ONE (2015)

Bottom Line: For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler.It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation.Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany; Hahn-Schickard, Freiburg, Germany.

ABSTRACT
Nested PCR remains a labor-intensive and error-prone biomolecular analysis. Laboratory workflow automation by precise control of minute liquid volumes in centrifugal microfluidic Lab-on-a-Chip systems holds great potential for such applications. However, the majority of these systems require costly custom-made processing devices. Our idea is to augment a standard laboratory device, here a centrifugal real-time PCR thermocycler, with inbuilt liquid handling capabilities for automation. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards. For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler. It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. 92.2% of the performed tests were recognized as fluidically failure-free sample handling and used for evaluation. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.

No MeSH data available.


Related in: MedlinePlus

Schematic description of a microfluidic disk segment (ā€œGeneSliceā€) with pre-stored reagents (A), and a compatible rotor holder (ā€œGeneSlice 100 Rotorā€), which can hold up to four GeneSlices in one Rotor-Gene Q run (B).The GeneSlice comprises pre-amplification chambers for sample and no-template control (NTC) liquids, capillary siphon valves for transfer of the pre-amplification product and a centrifugo-thermopneumatic two-stage aliquoting structure [31] for 14 (sample) and 1 (NTC) main-amplification(s), respectively. The aliquoting structures guide excess liquid into waste chambers. All required reagents are pre-stored in lyophilized or air-dried format. The air vent for pressure equalization is covered with a membrane. Siphon valves are rendered hydrophilic by coatings.
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pone.0131845.g001: Schematic description of a microfluidic disk segment (ā€œGeneSliceā€) with pre-stored reagents (A), and a compatible rotor holder (ā€œGeneSlice 100 Rotorā€), which can hold up to four GeneSlices in one Rotor-Gene Q run (B).The GeneSlice comprises pre-amplification chambers for sample and no-template control (NTC) liquids, capillary siphon valves for transfer of the pre-amplification product and a centrifugo-thermopneumatic two-stage aliquoting structure [31] for 14 (sample) and 1 (NTC) main-amplification(s), respectively. The aliquoting structures guide excess liquid into waste chambers. All required reagents are pre-stored in lyophilized or air-dried format. The air vent for pressure equalization is covered with a membrane. Siphon valves are rendered hydrophilic by coatings.

Mentions: The RGQ takes up rotors with tubes, which can either be spaced in 10Ā°, 5Ā°, or 3.6Ā° angular distances. A custom rotor holder (GeneSlice 100 Rotor) was designed (cf. Abstract Graphic), which provides pins to precisely align reaction cavities of the microfluidic disk segment, termed GeneSlice, to the stationary fluorescence detector of the RGQ while passing the detector under rotation (Fig 1B). The spacing of reaction cavities was set to 3.6Ā° to accommodate a maximum of parallel PCR reactions. The GeneSlice (Fig 1A) consists of two fluidic networks of chambers and channels fluidically processing one sample (left side) and the corresponding no-template control (NTC; right side) during nested PCR and subsequent melt curve analysis.


Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment.

Keller M, Naue J, Zengerle R, von Stetten F, Schmidt U - PLoS ONE (2015)

Schematic description of a microfluidic disk segment (ā€œGeneSliceā€) with pre-stored reagents (A), and a compatible rotor holder (ā€œGeneSlice 100 Rotorā€), which can hold up to four GeneSlices in one Rotor-Gene Q run (B).The GeneSlice comprises pre-amplification chambers for sample and no-template control (NTC) liquids, capillary siphon valves for transfer of the pre-amplification product and a centrifugo-thermopneumatic two-stage aliquoting structure [31] for 14 (sample) and 1 (NTC) main-amplification(s), respectively. The aliquoting structures guide excess liquid into waste chambers. All required reagents are pre-stored in lyophilized or air-dried format. The air vent for pressure equalization is covered with a membrane. Siphon valves are rendered hydrophilic by coatings.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492497&req=5

pone.0131845.g001: Schematic description of a microfluidic disk segment (ā€œGeneSliceā€) with pre-stored reagents (A), and a compatible rotor holder (ā€œGeneSlice 100 Rotorā€), which can hold up to four GeneSlices in one Rotor-Gene Q run (B).The GeneSlice comprises pre-amplification chambers for sample and no-template control (NTC) liquids, capillary siphon valves for transfer of the pre-amplification product and a centrifugo-thermopneumatic two-stage aliquoting structure [31] for 14 (sample) and 1 (NTC) main-amplification(s), respectively. The aliquoting structures guide excess liquid into waste chambers. All required reagents are pre-stored in lyophilized or air-dried format. The air vent for pressure equalization is covered with a membrane. Siphon valves are rendered hydrophilic by coatings.
Mentions: The RGQ takes up rotors with tubes, which can either be spaced in 10Ā°, 5Ā°, or 3.6Ā° angular distances. A custom rotor holder (GeneSlice 100 Rotor) was designed (cf. Abstract Graphic), which provides pins to precisely align reaction cavities of the microfluidic disk segment, termed GeneSlice, to the stationary fluorescence detector of the RGQ while passing the detector under rotation (Fig 1B). The spacing of reaction cavities was set to 3.6Ā° to accommodate a maximum of parallel PCR reactions. The GeneSlice (Fig 1A) consists of two fluidic networks of chambers and channels fluidically processing one sample (left side) and the corresponding no-template control (NTC; right side) during nested PCR and subsequent melt curve analysis.

Bottom Line: For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler.It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation.Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany; Hahn-Schickard, Freiburg, Germany.

ABSTRACT
Nested PCR remains a labor-intensive and error-prone biomolecular analysis. Laboratory workflow automation by precise control of minute liquid volumes in centrifugal microfluidic Lab-on-a-Chip systems holds great potential for such applications. However, the majority of these systems require costly custom-made processing devices. Our idea is to augment a standard laboratory device, here a centrifugal real-time PCR thermocycler, with inbuilt liquid handling capabilities for automation. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards. For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler. It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. 92.2% of the performed tests were recognized as fluidically failure-free sample handling and used for evaluation. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.

No MeSH data available.


Related in: MedlinePlus