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 sheep and goat amplimers.1 ng of sheep and goat DNA were amplified on two GeneSlices. The melt curve analysis shows the successful amplification of the 12S rRNA and cytb genes for both species. The different melting behavior is caused by inter-species sequence differences within the Caprinae.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131845.g003: Melt curve analysis of sheep and goat amplimers.1 ng of sheep and goat DNA were amplified on two GeneSlices. The melt curve analysis shows the successful amplification of the 12S rRNA and cytb genes for both species. The different melting behavior is caused by inter-species sequence differences within the Caprinae.

Mentions: 1 ng of DNA from two species of each animal group, except for Bovini, Homo sapiens and Sus scrofa, were used for verification of the animal-group-specific primers. A successful amplification was obtained in all cases by detection of the intended melt peaks for both mtDNA fragments (12S rRNA and cytb), as depicted for e.g. Caprinae in Fig 3. Macaque and budgerigar, not covered by the assay, only resulted in a positive amplification of the universal 12S rRNA fragment (DNA extraction control), whereas no signal was obtained for pogona and carp due to an insufficient amplification efficiency.


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 sheep and goat amplimers.1 ng of sheep and goat DNA were amplified on two GeneSlices. The melt curve analysis shows the successful amplification of the 12S rRNA and cytb genes for both species. The different melting behavior is caused by inter-species sequence differences within the Caprinae.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131845.g003: Melt curve analysis of sheep and goat amplimers.1 ng of sheep and goat DNA were amplified on two GeneSlices. The melt curve analysis shows the successful amplification of the 12S rRNA and cytb genes for both species. The different melting behavior is caused by inter-species sequence differences within the Caprinae.
Mentions: 1 ng of DNA from two species of each animal group, except for Bovini, Homo sapiens and Sus scrofa, were used for verification of the animal-group-specific primers. A successful amplification was obtained in all cases by detection of the intended melt peaks for both mtDNA fragments (12S rRNA and cytb), as depicted for e.g. Caprinae in Fig 3. Macaque and budgerigar, not covered by the assay, only resulted in a positive amplification of the universal 12S rRNA fragment (DNA extraction control), whereas no signal was obtained for pogona and carp due to an insufficient amplification efficiency.

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