Thesis
Development of a bead-based multiplexed PCR assay for the simultaneous detection of multiple Mycoplasma species
Washington State University
Master of Science (MS), Washington State University
2010
Handle:
https://hdl.handle.net/2376/103715
Abstract
We describe the development and analytical validation of a specific and sensitive 7-plex polymerase chain reaction assay coupled to a bead-based liquid suspension array for high throughput detection of multiple Mycoplasma spp. of ruminants. The assay employs a combination of newly designed and previously validated primer-probe sets that target genetic loci specific for Mycoplasma bovis, Mycoplasma mycoides cluster, Mycoplasma mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subspecies capripneumoniae (Mccp). PCR products were hybridized to specific oligonucleotide probes bound to spectrally unique, 5.6 µm diameter, carboxylated beads, and subsequently analyzed by flow cytometry. Analytical sensitivity for the targeted Mycoplasma species ranged from 10 fg to 1 pg of purified gDNA extracted from broth cultures (approximately 8-800 MmmSC genome equivalents). In silico comparison of primers and probes, and analytical assessment with a range of near-neighbor Mycoplasma species and multiple bacterial respiratory pathogens of ruminants demonstrated 100% analytical specificity of the assay. To assess assay performance and diagnostic specificity, 192 bovine respiratory samples were analyzed by incorporating a high throughput DNA extraction platform. The assay correctly classified all 192 field samples as negative for MmmSC or Mccp with an apparent 100% diagnostic specificity for these pathogens. 33/192 field samples were positive for M. bovis and all were confirmed by PCR or sequence verification of the uvrC gene. The results from this study indicate that the bead-based liquid suspension array will provide a reliable, analytically sensitive and specific platform to simultaneously interrogate ruminant respiratory samples for multiple Mycoplasma species, including Mycoplasma mycoides cluster organisms exotic to the United States. Sequential addition of primer-probe sets to the assay did not significantly impact analytical sensitivity of individual primer-probe combinations, suggesting that expanding the array to include more Mycoplasma species will not compromise overall performance.
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Details
- Title
- Development of a bead-based multiplexed PCR assay for the simultaneous detection of multiple Mycoplasma species
- Creators
- Daniel J. Righter
- Contributors
- Terry F. McElwain (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Veterinary Microbiology and Pathology, Department of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; Pullman, Wash. :
- Identifiers
- 99900525144701842
- Language
- English
- Resource Type
- Thesis