Real-time PCR as a novel approach to overcome problems in the diagnosis and differentiation of Brucella spp.

• Forschungsstelle: BLV
• Projektnummer: 1.06.02
• Projekttitel Englisch: Real-time PCR as a novel approach to overcome problems in the diagnosis and differentiation of Brucella spp.

Schlüsselwörter (Englisch)

Brucellosis, real-time PCR, diagnosis, MGB-probes.

Kurzbeschreibung (Englisch)

Switzerland is declared free of brucellosis since 1963. However, the occurrence of brucellosis in wild boar in Switzerland has been demonstrated. For this reason, a wild boar surveillance program was created, in order to obtain information about the disease status in wild boars, and to measure the risk of reintroduction of the disease.
The success of such a brucellosis surveillance project depends strongly on the reliability of the methods used for detection and identification of the bacteria. Currently, screening of brucellosis in animals is based on serological tests. These tests are indirect and can be unspecific due to cross reaction or subsensitive reactions in samples from areas with a low or subclinical prevalence of brucellosis (Bogdanovich et al., 2004). For differentiation of brucella species, restriction-fragment-length-polymorphisms (RFLP) technique targeting the omp2 locus is used. This test is time consuming, represent a risk of laboratory-acquired infections, and must be performed by highly skilled staff.
In this project, we propose the establishment and implementation of real-time PCR for detection and differen-tiation of brucella infections in animals, based on high sensitivity and specificity reported (Leal-Klevezas et al., 2000), speed, versatility in sample handling, and infection risk reduction of laboratory staff.

Projektziele (Englisch)

- Establishment of real-time PCR for detection of brucella genus in blood and organ samples of infected
animals (wild, zoo, and livestock animals).
- To complement the recommended wild boar surveillance, and simultaneously to compare sensitivity and
specificity of the conventional methods used with this of the real-time PCR.
- Based on fluorogenic MGB probe technology (Kutyavin et al., 2000), the third aim is to create species
specific probes for the establishment of a differential and multiplex real-time PCR. Real-time PCRs are
highly sensitive, for this reason we believe that bacterial isolation required for RFLP test can be avoided
after the establishment of the differential real-time PCR.

Publikationen / Ergebnisse (Deutsch)

29.10.2009: Umsetzungssitzung 2009: PCR ist etabliert, trotzdem keine grossen Fortschritt in der Brucella Diagnostik. Niedrige Sensitivität von PCR; ist evtl. nicht genügend. ZOBA vertraut anscheinend selbst nicht vollständig auf den PCR Test. Weitere Untersuchungen (Organe Kultur und PCR) folgen beim Fall Genf. Brucellen Diagnostik ist im Moment unbefriedigend. Neuer Status: „erledigt“ (mvo).

Publikationen / Ergebnisse (Englisch)

Hinic, V., et al. (2008) Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems. , J. Microbiol. Methods,  75: 2, 375-8. 

Hinic, V. (2007) Real-time PCR assay for the detection of Brucella spp. in naturally infected wild boars. Inaugural-Dissertation. Vetsuisse-Fakultät, Universität Bern.

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