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. 2020 Oct;9(10):e1105.
doi: 10.1002/mbo3.1105. Epub 2020 Aug 12.

Pentaplex real-time PCR for differential detection of Yersinia pestis and Y. pseudotuberculosis and application for testing fleas collected during plague epizootics

Ying Bai  1 , Vladimir Motin  2 , Russell E Enscore  1 , Lynn Osikowicz  1 , Maria Rosales Rizzo  1 , Andrias Hojgaard  1 , Michael Kosoy  3 , Rebecca J Eisen  1
Affiliations

Pentaplex real-time PCR for differential detection of Yersinia pestis and Y. pseudotuberculosis and application for testing fleas collected during plague epizootics

Ying Bai et al. Microbiologyopen. 2020 Oct.

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.

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Conflict of interest statement

The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions.

Figures

Figure 1
Figure 1
Amplification of DNA (100 pg/µl, 10 pg/µl, 1 pg/µl) with different primer concentrations ranging from 200 nM to 1000 nM. The amplification was grouped tightly by the concentration of DNA. The higher the DNA concentration, the earlier the amplification started (lower Ct value). Primer concentrations had no significant differences in the amplification efficiencies with a very small change of Ct value. The three arrows indicated the DNA concentration for each group was 100 pg/µl, 10 pg/µl, and 1 pg/µl from left to right. A. pst; B. caf1; C. ypo2088; D. opgG; E. 18S rRNA
Figure 2
Figure 2
Sensitivity testing. Amplification (left panel) and standard curve (right panel) of each target with DNA dilutions 2 pg, 1 pg, 0.5 pg, 0.2 pg, 0.1 pg, 50 fg, 20 fg, 10 fg, 5 fg, 2 fg, 1 fg, 0.5 fg, 0.2 fg, 0.1 fg, 0.05 fg, and 0.02 fg. Ct values were correlated to the DNA concentration for each target. A. pst; B. caf1; C. ypo2088; D. opgG; E. 18S rRNA
Figure 3
Figure 3
By performing the pentaplex real‐time PCR, one Oropsylla tuberculata flea collected from prairie dog burrows after a prairie dog die‐off in Wyoming in 2019 was positive to pst, caf1, ypo2088, and 18S rRNA, with Ct value 19.65, 21.08, 21.74, and 15.06, respectively, suggesting a local Y. pestis transmission
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