Skip to main content
ARS Home » Research » Publications at this Location » Publication #92788
Title: A 5' NUCLEASE PCR ASSAY TO DETECT YARSINIA PESTIS

Author
item HIGGINS, JAMES
item EZZELL, JOHN - FT DETRICK, MD
item HINNEBUSCH, B. - ROCKY MT. LAB., MONTANA
item SHIPLEY, MICHELLE - FT. DETRICK, MD
item HENCHAL, ERIK - FT. DETRICK, MD
item IBRAHIM, M. - FT. DETRICK, MD

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/13/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: Yersinia pestis is the causative agent of bubonic plague, a serious infectious disease. This bacterium can be transmitted by fleas. People, such as terrorists, foreign military forces, or extremists in the US, may attempt to use this agent in Biological Warfare against the United States. A quick and accurate technique is needed to allow soldiers to know if they are being exposed to any Y. pestis. The authors developed a polymerase chain reaction (PCR) assay that uses a unique "fluorogenic" reagent to detect DNA molecules from Y. pestis. The fluorogenic reagent allows the person performing the assay, to scan the PCR tubes using a laser. Tubes that have Y. pestis in them will emit light in response to the laser beam. Tubes that have other species of Yersinia, that are not good for Biological Warfare agents, will not emit light. The assay developed, can allow scientists and defense personnel to examine material for the presence of Y. pestis in as little as four to five hours. Old-fashioned methods involved spreading material on a petri dish, and waiting to see what grew on it after a day. It is possible to use the new assay to examine many samples simultaneously. The assay can now be adapted to use with novel, portably thermal cycler instruments, that operate on batteries and can be used out in the field. This means that it is no longer necessary to take samples back to a main laboratory to perform the assay.

Technical Abstract: The 5' nuclease polymerase chain reaction (PCR) assay uses a fluorescent-labelled oligonucleotide probe (TaqManu), to rapidly detect and quantitate DNA templates in clinical samples. We developed a 5' nuclease PCR assay targeting the pla locus of Yersinia pestis. The assay is species-specific, with a detection threshold of 2.1 x 105 copies of the pla target, or 1.6 pg of total cell DNA. The assay detected Y. pestis in experimentally infected Xenopsylla cheopis fleas, and in experimentally infected monkey blood and oropharyngeal swabs. The TaqMan assay was simple to perform, rapidly (less than 2.5 h) detected Y. pestis, and shows promise as a future field-adaptable technique.