Short communicationComparative performance of SNP typing and ‘Bruce-ladder’ in the discrimination of Brucella suis and Brucella canis
Introduction
The genus Brucella consists of a number of species that infect different mammalian hosts and can lead to serious disease in many domestic animals as well as being the cause of brucellosis, one of the most serious zoonotic diseases of man worldwide (Corbel, 2006). However, the genus is genetically conserved (Vizcaíno et al., 2000, Halling et al., 2005, Whatmore et al., 2007). This has been reflected in difficulties in identifying molecular markers for typing over the years. This is particularly the case for B. suis and B. canis (Moreno et al., 2002), which have long been considered particularly closely related (Ouahrani et al., 1993, Cloeckaert et al., 1995, Cloeckaert et al., 1996, Michaux-Charachon et al., 1997, Gandara et al., 2001, Whatmore et al., 2005).
Two novel assays have been described recently designed to rapidly type Brucella isolates to the species level. The first of these is a multiplex conventional PCR (‘Bruce-ladder’) exploiting unique differences in genomic structure by using an eight primer pair cocktail that produces specific PCR profiles for B. melitensis, B. ovis, B. abortus, B. suis, B. canis, B. neotomae, marine mammal Brucella, and the vaccine strains Rev1, RB51, and S19 (García-Yoldi et al., 2006). Routine use of this molecular typing tool in our own laboratory revealed that, although in general the assay performs excellently, there were problems with discrimination between B. canis and B. suis. This observation was confirmed recently when, during a large validation exercise, the assay was reported to have difficulty differentiating reliably between B. canis and B. suis (López-Goñi et al., 2008).
The second tool is a rapid real-time PCR based assay (Gopaul et al., 2008) that uses minor groove binding (MGB) protein probes that probe species-specific SNPs identified by extensive multilocus sequencing studies (Scott et al., 2007, Whatmore et al., 2007). Here we compare the performance of both these assays in typing a panel of Brucella isolates from dogs, previously identified as B. canis by phenotypic typing, and assess the basis of the inability of ‘Bruce-ladder’ to identify all B. canis isolates.
Section snippets
Strains
A panel of 24 B. canis isolates, collected from around the globe and typed using phenotypic typing (Alton et al., 1988), was examined in this study (Table 1). The template used for PCR consisted of crude extracts (cells killed by immersion in 66% (v/v) methanol or by boiling) or purified DNA obtained following phenol-chloroform extraction.
Molecular typing
Multilocus sequence analysis was carried out as described previously (Whatmore et al., 2007). The ‘Bruce-ladder’ PCR was performed according to the method of
Results
The basis for ‘Bruce-ladder’ differentiation between B. canis and B. suis is the amplification of a 794 bp fragment from B. suis, which is not amplified from B. canis because of the presence of a deletion encompassing the binding site of one of the primers (Rajashekara et al., 2004). The ‘Bruce-ladder’ multiplex PCR (García-Yoldi et al., 2006) was tested against the strains indicated in Table 1, all previously identified as B. canis by standard microbiological procedures (Alton et al., 1988).
Discussion
Both ‘Bruce-ladder’ and the SNP based assay were used to examine a panel of strains confirmed as B. canis by phenotypic typing. Both assays are PCR based and were found to be simple and reproducible tools that could readily be used in routine diagnostics. ‘Bruce-ladder’ has the advantage of being a single tube assay while SNP typing requires a set-up of a number of individual reactions, although the procedure is simple as all are optimised such that they are carried out simultaneously on a
Acknowledgement
Brucella research at the VLA is supported by the UK Department of the Environment, Food and Rural Affairs (Defra).
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