New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis
Introduction
Brucella suis, the causative agent of swine brucellosis, is classified in five biovars that infect different animal hosts: biovars 1, 2 and 3 affect domestic pigs, wild boars and hares; biovar 4 infects reindeer and caribou; and biovar 5 infects only rodents. In contrast to biovar 2, biovars 1 and 3 are pathogenic for humans and require high level of biosafety laboratory precautions (Garin-Bastuji and Hars, 2001). The identification and typing of B. suis is currently performed by standard bacteriological and biochemistry methods, but these tests are not straightforward in particular for the identification of biovars 1, 2 and 3 (Alton et al., 1988). With the objective of improving the typing of B. suis, different PCR-based assays have been proposed. One of these assays is a multiplex conventional PCR (Bruce-ladder) (García-Yoldi et al., 2006, López-Goñi et al., 2008). In general Bruce-ladder performs excellently and has been recommended by the OIE as a rapid and simple one-step molecular test for identification and typing of Brucella species (OIE, 2009). However, its only inconvenience is that some Brucella canis strains and Brucella microti, isolated from common voles and foxes, can be identified erroneously as B. suis (López-Goñi et al., 2008, Scholz et al., 2008). The present report describes a novel multiplex conventional PCR assay (called Suis-ladder) that differentiates between all B. suis biovars and B. canis, and also propose an advancement of the original Bruce-ladder PCR to distinguish between these two species.
Section snippets
Strains
To ensure an adequate diversity, a representative collection of B. suis and B. canis reference strains and field isolates from different geographic origins and different animal species was examined (see supplemental material). When needed, Brucella reference strains were used. All Brucella isolates were typed according to standard procedures (Alton et al., 1988). Growth and harvesting of Brucella cells and bacterial DNA extraction were performed as described elsewhere (García-Yoldi et al., 2006
Results and discussion
A representative example of the multiplex Suis-ladder PCR result is presented in Fig. 1. PCR using B. suis DNAs from the five reference biovars gave different band profiles: biovar 1 amplified three fragments of 774, 425 and 197 bp; biovar 2 amplified three fragments of 774, 551 and 278 bp; biovar 3 amplified three fragments of 774, 299 and 197 bp; biovar 4 amplified three fragments of 774, 614 and 197 bp; and biovar 5 amplified four fragments of 774 bp, 614, 278 and 197 bp. Then, the Suis-ladder PCR
Acknowledgements
This work was partially supported by Ministerio de Educación y Ciencia, Spain (AGL2008-04514, PET2008-0027 and FAU2008-00015). We want to express our gratitude to Pilar Mª Muñoz, Zeljko Cvetnic, David Fretin, Maria Silvia Gennero, Falk Melzer, R. Miserez, Lorraine Perrett, and Isabel Travassos for providing some of the Brucella isolates.
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