Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

Abstract

Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.Brucella spp. are pathogenic bacteria that infect a wide variety of mammalian hosts worldwide, often causing reproductive failure. The genus Brucella has classically been divided into six species based on host specificity, including B. abortus (cattle and bison), B. melitensis (goats and sheep), B. suis (pigs), B. canis (dogs), B. neotomae (desert woodrat), and B. ovis (sheep) (12). Two new species have been discovered recently in marine mammals (B. cetaceae in dolphins and whales and B. pinnipediae in seals) (10). Taxonomic limits of the marine clade, however, are not fully defined, and this group may represent one to three species (8, 18). B. abortus, B. melitensis, B. suis, and B. canis are well-characterized zoonotic pathogens, annually infecting Ͼ500,000 people worldwide (26). In the United States, the first three of these species are defined as select agents due to their pathogenicity and potential use as biological weapons (11).Despite host-based segregation, Brucella spp. have proven challenging to differentiate using molecular techniques. Brucella genomes are highly conserved, with Ͼ90% homology among species based on DNA-DNA hybridization (35), identical 16S rRNA sequences among all species (15), and Ͼ90% of genes sharing Ͼ98% sequence identity (16,27). Serological methods and biochemical testing of isolates allow differentiation of species and biovars. However, PCR-based methods have been used increasingly due to their accuracy, sensitivity, and speed of identification and the ability to work with DNA as opposed to highly infectious live cultures. A wide array of genetic polymorphisms can be assayed for the differentiation of Brucella spp., including the insertion element IS711 (2, 3, 29, 31) and genes of outer membrane proteins (7,...

Development of a PCR assay for typing and subtyping of Brucella species

“…Concerning sensitivity and rapidness, a conventional PCR like our multiplex PCR assay cannot compete with a real-time PCR as developed by Foster et al (2008) for specific detection of 7 major Brucella clades. However, for identification of a sample of unknown source, 7 different real-time PCRs have to be run in parallel to reach this goal.…”

“…However, for identification of a sample of unknown source, 7 different real-time PCRs have to be run in parallel to reach this goal. Furthermore, the approach of Foster et al (2008) does not distinguish between biovars of certain species or between the marine brucellae according to current taxonomy, nor does it identify B. microti.…”

Extended Multilocus Sequence Analysis to Describe the Global Population Structure of the Genus Brucella: Phylogeography and Relationship to Biovars

“…Given advances in molecular approaches it would seem timely to consider whether such approaches offer a more meaningful replacement for biotyping. Such assays could be based on genomic rearrangements such as the multiplex Bruceladder (López-Goñi et al, 2008) or SNPs as per existing speciation assays (Scott et al, 2007; Foster et al, 2008; Gopaul et al, 2008) which have the advantage that they can be applied at a number of taxonomic levels (Keim et al, 2004). The crucial point is that they must be designed on the back of a robust population genetic understanding provided by data such as that presented here and iteratively re-examined as knowledge of extant diversity increases.…”

“…Further this traditional method is likely to become increasingly less relevant as the genus expands and new species emerge that diverge from classical criteria. As more molecular diversity has become apparent with technological advances biotyping is increasingly being replaced by the use of frontline molecular tools notably various diagnostic PCRs based on genomic deletions or SNPs (Foster et al, 2008; Gopaul et al, 2008, 2010; López-Goñi et al, 2008). However the performance of such tools is critically dependent on their design being based on an accurate and comprehensive understanding of the population genetic structure of the groups they are designed to separate (Keim et al, 2004).…”

Imported human brucellosis in Belgium: Bio and molecular typing of bacterial isolates, 1996-2015

“…In some cases, appearance of mutants that generate genetic diversity is responsible for atypical susceptibility to dyes or delayed agglutination with anti- Brucella sera and thus hinders an accurate identification of the species and biovar [12]. Molecular genotyping based on single gene or multilocus sequence typing to identify locus polymorphisms or other mutations in genes have also been developed to understand the phylogeny of the genus Brucella and try to address the question of species and biovars [25][26]. These methods are not exhaustive when sub-typing for epidemiological trace-back is necessary.…”