@article{ca5743fb58a64820b6c55db11b92f1f4,
title = "Pentaplex real-time PCR for differential detection of Yersinia pestis and Y. pseudotuberculosis and application for testing fleas collected during plague epizootics",
abstract = "Upon acquiring two unique plasmids (pMT1 and pPCP1) and genome rearrangement during the evolution from Yersinia pseudotuberculosis, the plague causative agent Y. pestis is closely related to Y. pseudotuberculosis genetically but became highly virulent. We developed a pentaplex real-time PCR assay that not only detects both Yersinia species but also differentiates Y. pestis strains regarding their plasmid profiles. The five targets used were Y. pestis-specific ypo2088, caf1, and pst located on the chromosome, plasmids pMT1 and pPCP1, respectively; Y. pseudotuberculosis-specific chromosomal gene opgG; and 18S ribosomal RNA gene as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 fg to 100 fg, with Y. pestis-specific pst as the most sensitive target. Using the assay, Y. pestis strains were differentiated 100% by their known plasmid profiles. Testing Y. pestis and Y. pseudotuberculosis-spiked flea DNA showed there is no interference from flea DNA on the amplification of targeted genes. Finally, we applied the assay for testing 102 fleas collected from prairie dog burrows where prairie dog die-off was reported months before flea collection. All flea DNA was amplified by 18S rRNA; no Y. pseudotuberculosis was detected; one flea was positive for all Y. pestis-specific targets, confirming local Y. pestis transmission. Our results indicated the assay is sensitive and specific for the detection and differentiation of Y. pestis and Y. pseudotuberculosis. The assay can be used in field investigations for the rapid identification of the plague causative agent.",
keywords = "Yersinia pestis, Yersinia pseudotuberculosis, assay development, fleas, pentaplex real-time PCR",
author = "Ying Bai and Vladimir Motin and Enscore, {Russell E.} and Lynn Osikowicz and {Rosales Rizzo}, Maria and Andrias Hojgaard and Michael Kosoy and Eisen, {Rebecca J.}",
note = "Funding Information: The following reagents were obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: Genomic DNA from Yersinia pestis, Strain KIM10+, NR-2645; Genomic DNA from Yersinia pestis, Strain PB, NR-2718; Genomic DNA from Yersinia pestis, Strain Harbin 35, NR-2719; Genomic DNA from Yersinia pestis, Strain Nepal 516, NR-2720; Genomic DNA from Yersinia pestis, Strain KIM Derivative 19 (D19), NR-4705; Genomic DNA from Yersinia pestis, Strain KIM Derivative 2 (D2), NR-4706; Genomic DNA from Yersinia pestis, Strain KIM Derivative 22 (D22), NR-4708; Genomic DNA from Yersinia pestis, Strain KIM Derivative 22 (D22), NR-4708; Genomic DNA from Yersinia pestis, Strain A12 Derivative 6 (D6), NR-4713; Genomic DNA from Yersinia pestis, Strain Kuma Derivative 7 (D7), NR-4714; Genomic DNA from Yersinia pestis, Strain yokohama Derivative 10 (D10), NR-4716; Genomic DNA from Yersinia pestis, Strain yokohama Derivative 11 (D11), NR-4717; Genomic DNA from Yersinia pestis, Strain Kimberley Derivative 12 (D12), NR-4718; Genomic DNA from Yersinia pestis, Strain Kimberley Derivative 13 (D13), NR-4719; Genomic DNA from Yersinia pestis, Strain K25 Derivative 72 (D72), NR-4721; Genomic DNA from Yersinia pestis, Strain K25 Derivative 80 (D80), NR-4727;; Genomic DNA from Yersinia pseudotuberculosis, Strain IP2775, NR-4651; Genomic DNA from Yersinia pseudotuberculosis, Strain YPIII(p+), NR-4653; Genomic DNA from Yersinia enterocolita, Strain Billups-1803-68, NR-3064; and Genomic DNA from Yersinia enterocolita, Strain Billups-1803-68, NR-3064. We thank Scott Bearden from the Bacterial Disease Branch, Division of Vector-Borne Diseases, Centers for Disease Control and Prevention for providing genomic DNA from Yersinia pestis, strain CO96-3166 and genomic DNA from Yersinia pseudotuberculosis, strain B15. We thank Nicole Breuner, Erik Foster, Christina Parise, Sarah Maes, Aine Lehane, Christine Graham, Amy Fleshman, and Karen Boegler from the Bacterial Disease Branch, Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, and Daniel O{\textquoteright}Leary, Wyoming Department of Health, for their participation in the field collection of fleas. Publisher Copyright: {\textcopyright} 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd",
year = "2020",
month = oct,
day = "1",
doi = "10.1002/mbo3.1105",
language = "English (US)",
volume = "9",
journal = "MicrobiologyOpen",
issn = "2045-8827",
publisher = "John Wiley and Sons Inc.",
number = "10",
}