Journal article
Development of a bead-based multiplex PCR assay for the simultaneous detection of multiple Mycoplasma species
Veterinary microbiology, Vol.153(3), pp.246-256
2011
Handle:
https://hdl.handle.net/2376/107499
PMID: 21726966
Abstract
We describe the development and analytical validation of a 7-plex polymerase chain reaction assay coupled to a bead-based liquid suspension array for detection of multiple ruminant
Mycoplasma spp. 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). 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 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 samples as negative for MmmSC or Mccp. All 33 field samples confirmed as positive for
M. bovis by sequencing the
uvrC gene were positive in the assay. 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
M. mycoides cluster organisms that are 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 assay to include more
Mycoplasma species will not compromise overall performance.
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Details
- Title
- Development of a bead-based multiplex PCR assay for the simultaneous detection of multiple Mycoplasma species
- Creators
- Daniel J Righter - Washington Animal Disease Diagnostic Laboratory, PO Box 647040, Washington State University, Pullman, WA 99164-7040, United StatesFred R Rurangirwa - Washington Animal Disease Diagnostic Laboratory, PO Box 647040, Washington State University, Pullman, WA 99164-7040, United StatesDouglas R Call - Department of Veterinary Microbiology and Pathology, PO Box 647040, Washington State University, Pullman, WA 99164-7040, United StatesTerry F McElwain - Washington Animal Disease Diagnostic Laboratory, PO Box 647040, Washington State University, Pullman, WA 99164-7040, United States
- Publication Details
- Veterinary microbiology, Vol.153(3), pp.246-256
- Academic Unit
- Paul G. Allen School for Global Animal Health
- Publisher
- Elsevier B.V
- Identifiers
- 99900547257901842
- Language
- English
- Resource Type
- Journal article