Application of chromosomal DNA and protein targeting for the identification of Yersinia pestis

Purpose: A comprehensive strategy was developed and validated for the identification of pathogens from closely related near neighbors using both chromosomal and protein biomarkers, with emphasis on distinguishing Yersinia pestis from the ancestral bacterium Yersinia pseudotuberculosis. Experimental design: Computational analysis was used to discover chromosomal targets unique to Y. pestis. Locus identifier YPO1670 was selected for further validation and PCR was used to confirm that this biomarker was exclusively present in Y. pestis strains, while absent in other Yersinia species. RT-PCR and Western blot analyses were utilized to evaluate YPO1670 expression and MRM MS was performed to identify the YPO1670 protein within cell lysates. Results: The described study validated that YPO1670 was exclusive to Y. pestis .P CR confirmed the locus to be unique to Y. pestis. The associated transcript and protein were produced throughout growth with the highest abundance occurring in stationary phase and MRM MS conclusively identified the YPO1670 protein in cell extracts. Conclusions and clinical relevance: These findings validated YPO1670 as a reliable candidate biomarker for Y. pestis and that a dual DNA and protein targeting approach is feasible for the development of next-generation assays to accurately differentiate pathogens from near