Development of a rapid viability polymerase chain reaction method for detection of Yersinia pestis

Publication date: Available online 13 May 2019Source: Journal of Microbiological MethodsAuthor(s): Staci R. Kane, Sanjiv R. Shah, Teneile M. AlfaroAbstractDue to the occurrence of natural plague outbreaks and its historical usage as a biological weapon, Yersinia pestis is considered one of the high-priority biological threat agents. It can remain viable in certain environments including water for>100 days. Because of its slow-growth characteristic, it usually takes three or more days to detect and confirm the identity of viable Y. pestis cells by PCR, serological, or biochemical assays when using the traditional microbiological plate-culture-based analysis, and that too, assuming faster growing microbes present in a water sample do not mask the Y. pestis colonies and interfere with analysis. Therefore, a rapid-viability Polymerase Chain Reaction (RV-PCR) method was developed for detection of Y. pestis. The RV-PCR method combines 24 h-incubation broth culture in a 48-well plate, and pre- and post-incubation differential PCR analyses, thereby allowing for rapid and high-throughput sample analysis compared with the current plate culture method. One chromosomal and two plasmid gene target-based real-time PCR assays were down-selected, showing ca. 10 genome equivalent detection; the chromosomal assay was then used for RV-PCR method development. A 101-cell level (10–99 cells) sensitivity of detection was demonstrated even with complex sample backgrounds including known PCR in...
Source: Journal of Microbiological Methods - Category: Microbiology Source Type: research