|Del Vecchio, Vito|
Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/7/2000
Publication Date: N/A
Citation: Interpretive Summary: Brucellosis is an infectious disease of livestock and other animals. It occurs worldwide in cattle, sheep, goats, swine and dogs, as well as bison, elk, camels, reindeer and marine mammals. Because of the serious impact on agriculture, the USA has nearly eradicated brucellosis from cattle and is working towards its eradication from swine, bison and elk. All market cattle are tested for exposure to the disease. Previously, scientists at the NADC were successful in developing a test that could identify the disease-causing bacteria in a single day, compared to the usual period of approximately 2 weeks. The APHIS Diagnostic Lab in Ames, Iowa now routinely uses this test as a preliminary screen. The work presented in the current paper now reduces that time period from a full day to less than an hour. The results can be monitored while the test is performed. This rapid turn around of results could generate significant savings to affected producers.
Technical Abstract: Real-time PCR-based assays specific for Brucella abortus, B. melitensis and B. suis were developed. The assays utilize an upstream primer that is derived from 3' end of the genetic element IS711, whereas the downstream primers and probes are designed from signature sequences specific to a species or a biovar. The PCR reactions were monitored for fluorescence resonance energy transfer by including two adjacent labeled probes that hybridize to the amplicons as they are formed. The upstream probes were labeled with fluorescein at 3' end while Cy5 was attached to the 5' end of the downstream probes. An increase in the ratio of fluorescein to Cy5 fluorescence during the cycling was indicative of positive amplification event. The assays were accomplished in less than 30 min using a LightCycler in real-time mode. The assays were tested on known strains as well as field isolates and were found to be specific for all known biovars of B. abortus, B. melitensis and biovar 1 of B. suis. Thus specificity, sensitivity, speed and real-time detection make these assays attractive for use in epidemiological and ecological studies.