دورية أكاديمية
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.
| العنوان: | 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. |
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| المؤلفون: | Mark Keller, Jana Naue, Roland Zengerle, Felix von Stetten, Ulrike Schmidt |
| المصدر: | PLoS ONE, Vol 10, Iss 7, p e0131845 (2015) |
| بيانات النشر: | Public Library of Science (PLoS), 2015. |
| سنة النشر: | 2015 |
| المجموعة: | LCC:Medicine LCC:Science |
| مصطلحات موضوعية: | Medicine, Science |
| الوصف: | 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. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 1932-6203 |
| العلاقة: | http://europepmc.org/articles/PMC4492497?pdf=renderTest; https://doaj.org/toc/1932-6203Test |
| DOI: | 10.1371/journal.pone.0131845 |
| الوصول الحر: | https://doaj.org/article/fd1fa68be7e442e8b0c78a549a40e046Test |
| رقم الانضمام: | edsdoj.fd1fa68be7e442e8b0c78a549a40e046 |
| قاعدة البيانات: | Directory of Open Access Journals |
Full Text Finder | تدمد: | 19326203 |
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| DOI: | 10.1371/journal.pone.0131845 |