Limits...
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

Melt curve analysis of a mixture of human and pig DNA in different ratios.Artificial mixtures of human and pig DNA were created from 0% to 100%, each, and amplified. Analysis of the respective animal-group-specific main-amplification and universal 12S rRNA cavities shows resolution of the DNA components down to 1% (12S rRNA) for pig and human; 5% both for pig, and 10% both for human as minor components.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492497&req=5

pone.0131845.g004: Melt curve analysis of a mixture of human and pig DNA in different ratios.Artificial mixtures of human and pig DNA were created from 0% to 100%, each, and amplified. Analysis of the respective animal-group-specific main-amplification and universal 12S rRNA cavities shows resolution of the DNA components down to 1% (12S rRNA) for pig and human; 5% both for pig, and 10% both for human as minor components.

Mentions: Mixing of human and pig DNA (1 ng in total) led to successful detection of human and pig down to 1% for 12S rRNA as well as 5% for pig cytb and 10% for human cytb (Fig 4). In all cases, the included internal positive control (IPC) and the universal 12S rRNA used as positive DNA control showed the expected results. The IPC showed a stable melting behavior with a melting temperature of 81.71 ± 0.23°C over all measurements. There were only small deviations within the triplicates, with a maximum ± 0.13°C shift of Tm for the visually detectable universal, human-specific and pig-specific melting peaks.


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)

Melt curve analysis of a mixture of human and pig DNA in different ratios.Artificial mixtures of human and pig DNA were created from 0% to 100%, each, and amplified. Analysis of the respective animal-group-specific main-amplification and universal 12S rRNA cavities shows resolution of the DNA components down to 1% (12S rRNA) for pig and human; 5% both for pig, and 10% both for human as minor components.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131845.g004: Melt curve analysis of a mixture of human and pig DNA in different ratios.Artificial mixtures of human and pig DNA were created from 0% to 100%, each, and amplified. Analysis of the respective animal-group-specific main-amplification and universal 12S rRNA cavities shows resolution of the DNA components down to 1% (12S rRNA) for pig and human; 5% both for pig, and 10% both for human as minor components.
Mentions: Mixing of human and pig DNA (1 ng in total) led to successful detection of human and pig down to 1% for 12S rRNA as well as 5% for pig cytb and 10% for human cytb (Fig 4). In all cases, the included internal positive control (IPC) and the universal 12S rRNA used as positive DNA control showed the expected results. The IPC showed a stable melting behavior with a melting temperature of 81.71 ± 0.23°C over all measurements. There were only small deviations within the triplicates, with a maximum ± 0.13°C shift of Tm for the visually detectable universal, human-specific and pig-specific melting peaks.

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