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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

Illustration of microfluidic process flow of a GeneSlice for nested PCR and melt curve analysis on a Rotor-Gene Q.The microfluidic structures of interest at different points in time (A-E) are shown on top, indicating liquid movement from the dark blue to the light blue position. Corresponding temperatures (red) and rotational speed (black) at each depicted point in time can be read from the given diagram at the bottom. The liquid stays in the position depicted in B during pre-amplification, which consists of 10 thermal PCR cycles at constant 400 RPM. During main-amplification and melt curve analysis, which follow the last (E) depicted fluidic operation, no further fluidic operations take place and liquids stay in the main-amplification cavities (E, light blue).
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pone.0131845.g002: Illustration of microfluidic process flow of a GeneSlice for nested PCR and melt curve analysis on a Rotor-Gene Q.The microfluidic structures of interest at different points in time (A-E) are shown on top, indicating liquid movement from the dark blue to the light blue position. Corresponding temperatures (red) and rotational speed (black) at each depicted point in time can be read from the given diagram at the bottom. The liquid stays in the position depicted in B during pre-amplification, which consists of 10 thermal PCR cycles at constant 400 RPM. During main-amplification and melt curve analysis, which follow the last (E) depicted fluidic operation, no further fluidic operations take place and liquids stay in the main-amplification cavities (E, light blue).

Mentions: Starting the pre-amplification, the loaded sample/NTC liquids are transferred from their loading areas into the radially most outward position of the pre-amplification chambers by constant centrifugation of 400 RPM (Fig 2A). The first capillary valves (Fig 2, I) directly break by the centrifugal pressure of the sample/NTC liquid. Breaking of the valves is enhanced by the lowered surface tension and degassing of liquid, which pushes liquid through the valve. Both is caused by the temperature ramp up to the hot start temperature of the polymerase (Fig 2B). During further constant centrifugation, the liquids stay in the pre-amplification chambers, and pre-amplification of 10 PCR cycles is carried out. Afterward, a programmed cool-down to 30°C takes place (hold at 40°C for 0 s, at 35°C for 0 s, at 30°C for 40 s), before the RGQ shuts down resulting in a cool-down to room temperature and a subsequent halt of rotation. During this resting period, the capillary pressures inside the hydrophilic siphon valves pull the pre-amplification products beyond the radially inward siphon’s crests to the capillary valves (Fig 2, II), which stop further capillary priming (Fig 2C) [17]. When the main-amplification is initiated, the rotor accelerates to 400 RPM and the centrifugal pressures break the capillary valves (Fig 2, II). The liquids are propelled downstream into the corresponding CTP two-stage aliquoting structures.


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)

Illustration of microfluidic process flow of a GeneSlice for nested PCR and melt curve analysis on a Rotor-Gene Q.The microfluidic structures of interest at different points in time (A-E) are shown on top, indicating liquid movement from the dark blue to the light blue position. Corresponding temperatures (red) and rotational speed (black) at each depicted point in time can be read from the given diagram at the bottom. The liquid stays in the position depicted in B during pre-amplification, which consists of 10 thermal PCR cycles at constant 400 RPM. During main-amplification and melt curve analysis, which follow the last (E) depicted fluidic operation, no further fluidic operations take place and liquids stay in the main-amplification cavities (E, light blue).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131845.g002: Illustration of microfluidic process flow of a GeneSlice for nested PCR and melt curve analysis on a Rotor-Gene Q.The microfluidic structures of interest at different points in time (A-E) are shown on top, indicating liquid movement from the dark blue to the light blue position. Corresponding temperatures (red) and rotational speed (black) at each depicted point in time can be read from the given diagram at the bottom. The liquid stays in the position depicted in B during pre-amplification, which consists of 10 thermal PCR cycles at constant 400 RPM. During main-amplification and melt curve analysis, which follow the last (E) depicted fluidic operation, no further fluidic operations take place and liquids stay in the main-amplification cavities (E, light blue).
Mentions: Starting the pre-amplification, the loaded sample/NTC liquids are transferred from their loading areas into the radially most outward position of the pre-amplification chambers by constant centrifugation of 400 RPM (Fig 2A). The first capillary valves (Fig 2, I) directly break by the centrifugal pressure of the sample/NTC liquid. Breaking of the valves is enhanced by the lowered surface tension and degassing of liquid, which pushes liquid through the valve. Both is caused by the temperature ramp up to the hot start temperature of the polymerase (Fig 2B). During further constant centrifugation, the liquids stay in the pre-amplification chambers, and pre-amplification of 10 PCR cycles is carried out. Afterward, a programmed cool-down to 30°C takes place (hold at 40°C for 0 s, at 35°C for 0 s, at 30°C for 40 s), before the RGQ shuts down resulting in a cool-down to room temperature and a subsequent halt of rotation. During this resting period, the capillary pressures inside the hydrophilic siphon valves pull the pre-amplification products beyond the radially inward siphon’s crests to the capillary valves (Fig 2, II), which stop further capillary priming (Fig 2C) [17]. When the main-amplification is initiated, the rotor accelerates to 400 RPM and the centrifugal pressures break the capillary valves (Fig 2, II). The liquids are propelled downstream into the corresponding CTP two-stage aliquoting structures.

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