Pentaplex real‐time PCR for differential detection of Yersinia pestis and Y. pseudotuberculosis and application for testing fleas collected during plague epizootics. Issue 10 (12th August 2020)

• Record Type: Journal Article
• Authors: Bai, Ying; Motin, Vladimir; Enscore, Russell E.; Osikowicz, Lynn; Rosales Rizzo, Maria; Hojgaard, Andrias; Kosoy, Michael; Eisen, Rebecca J.
• Is Part Of: MicrobiologyOpen. Volume 9:Issue 10(2020)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-08-12
• Subjects: assay development -- fleas -- pentaplex real‐time PCR -- Yersinia pestis -- Yersinia pseudotuberculosis; Microbiology -- Periodicals; 579
• DOI: 10.1002/mbo3.1105
• Languages: English
• ISSNs: 2045-8827

Abstract

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. Abstract : We developed a pentaplex real‐time PCR assay including Yersinia pestis ‐specific genes ypo2088, caf1, and pst located on the chromosome, as well as on plasmids pMT1 and pPCP1, respectively. Yersinia pseudotuberculosis ‐specific chromosomal gene opgG and the 18S ribosomal RNA gene served as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 to 100 femtograms. Using the assay, Y. pestis was detected in field‐collected fleas and strains were differentiated 100% by their known plasmid profiles.