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Definitive identification of Brucella species remains a challenge due to the high degree of genetic homology shared within the genus. We report the development of a molecular technique which utilizes real-time PCR followed by high-resolution melt (HRM) curve analysis to reliably type members of this genus. Using a panel of seven primer sets, we tested 153 Brucella spp. isolates with >99% accuracy compared to traditional techniques. This assay provides a useful diagnostic tool that can rapidly type Brucella isolates and has the potential to detect novel species. This approach may also prove helpful for clinical, epidemiological and veterinary investigations.
The Brucella genus is composed of nine recognized species, six of which are classical members (Brucella abortus, B. suis, B. melitensis, B. canis, B. ovis, and B. neotomae), along with newly designated species B. ceti, B. pinnipedialis, and B. microti and the proposed species B. inopinata (5, 6, 15, 16). These small, facultative intracellular, aerobic, Gram-negative coccobacilli have a worldwide distribution and share over 90% nucleic acid sequence homology (19). As the causative agent of brucellosis, a zoonotic disease resulting in high rates of reproductive failure and sterility in livestock, these agents are most commonly transmitted to humans through the consumption of unpasteurized milk products, direct contact with infectious animal tissues, or inhalation of aerosolized droplets (13). B. melitensis, B. suis, B. abortus, and rarely B. canis are responsible for over 500,000 human infections per year worldwide, with the first three species classified as select agents due to their potential for bioterrorism (4, 5). Members of this genus also pose a significant risk for laboratory-acquired infections.
Historically, the Brucelleae were named according to their host preference, while taxonomy and species discrimination relied on biochemical, antigenic, and metabolic differences. Conventional laboratory tests such as CO2 requirement, dye sensitivities, phage susceptibility, H2S production, oxidative metabolic patterns, and antiserum reactivity are used to determine phenotypic characteristics in order to identify an isolate (20). These tests are often laborious and time-consuming, pose a risk of infection, and can generate discordant results. Recent molecular approaches such as whole genome sequence comparisons, single nucleotide polymorphism (SNP) analysis, multilocus variable-number tandem-repeat (VNTR) analysis (MLVA), and various real-time PCR approaches have greatly enhanced the ability to rapidly characterize members of this genus (1, 3, 7, 11, 17, 18, 21). Although these methodologies have proved greatly beneficial to the study of this genus, the assay described here provides an alternative approach to further delineate and augment the ability to rapidly identify these agents.
We report the development of a real-time PCR assay that utilizes high-resolution melt (HRM) analysis to specifically detect and discriminate members of this genus. HRM is a relatively new technology that allows for superb resolution and characterization of amplified nucleic acid targets by using a precisely regulated melting temperature profile that provides exceptional sensitivity for discerning minor differences (22). The major aim of the present study was to design a rapid and simple molecular assay capable of specifically detecting and discriminating major species of the genus and yet have the potential to identify unusual or novel isolates by exploiting the inherent advantages of HRM technology. To our knowledge, this is the first report to demonstrate the utility of this technology for characterizing this genus. The assay described here uses PCR amplification of seven independent loci, followed by dissociation curve analysis with five of the target regions, allowing multiple targets to be concurrently evaluated to make a positive species determination. Lastly, this assay may be useful for detecting Brucella spp. in clinical specimens and provide insight into epidemiological, clinical, and veterinary studies.
A total of 153 Brucella strains (representing eight species) from a collection of more than 1,000 were selected to represent temporal, geographic, and source diversity (Table (Table1).1). These isolates were obtained from 1973 to the present and represent a broad geographic distribution (the United States [including Hawaii], Europe, Puerto Rico, China, Mexico, Egypt, and Australia). A wide variety of sources were also represented, including blood, tissue, and fluids from humans, marine mammals, cattle, dogs, sheep, and rodents. All strains were stored at −70°C in defibrinated rabbit blood until testing. Identification of all strains was carried out using standard microbiological procedures as previously described (20). Bacteria were grown by plating one loop (1 μl) of stock cell suspension on Trypticase soy agar with 5% sheep blood agar (BBL Microbiology Systems, Cockeysville, MD) and incubating the bacteria aerobically for 1 to 2 days at 37°C with 5% CO2. DNA template was prepared as previously described (8).
Primers were manually designed to amplify selected regions for species determination by using publically available sequence data within the NCBI database. The primer designation, sequence, targeted loci used, and specificity for each marker are listed in Table Table2.2. Briefly, targeted sequences for each marker were aligned using CLUSTAL W, and primers were designed to provide discriminating HRM profiles. The marker specificities were as follows: Bspp, all Brucella spp.; Bmel, B. melitensis; Bcan, B. canis; Bmar, marine strains (B. ceti and B. pinnipedialis); Bneo, B. neotomae; Boa, B. ovis and B. abortus; and Bsui, B. suis. The real-time PCR assay was prepared using a Universal SYBR GreenER qPCR kit (Invitrogen, Carlsbad, CA) containing the following components per reaction: 12.5 μl of 2× master mix, a 100 nM final concentration of the forward and reverse primers, ~2 ng of template, and nuclease-free water (Promega, Madison, WI) to a total reaction volume of 25 μl. The real-time PCR was performed in triplicate on a Corbett Rotor-Gene 6000 (Qiagen, Valencia, CA) with the following run conditions: 1 cycle of 50°C for 2 min and 1 cycle of 95°C for 10 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s, with data acquired at the 60°C step in the green channel. After amplification, a high-resolution melt was performed between 73 and 88°C at a rate of 0.03°C per step. HRM curves for five markers were normalized using the specific temperature regions listed in Table Table22 prior to performing final analysis for reasons previously described (23). A positive/negative amplification growth curve only is required for the genus-specific marker (Bspp) and the B. suis-specific marker (Bsui).
A panel of 16 closely related non-Brucella species (n = 31) and human DNA was selected to test the specificity of the assay. These includes Ochrobactrum anthropi 5D (n = 4), Ochrobactrum intermedium, Agrobacterium radiobacter (n = 2), Agrobacterium tumefaciens, Agrobacterium radiobacter (n = 2), Oligella urethralis (n = 4), Afipia felis, Afipia broomeae, Haemophilus influenzae (n = 2), Psychrobacter phenylpyruvicus, Escherichia coli (n = 2), Salmonella enterica serovar Typhi (n = 3), Vibrio cholerae (n = 2), Yersinia enterocolitica (n = 3), Mycoplana spp., and Rhizobium spp. These were tested at 20 to 50 ng per reaction in duplicate.
The sensitivity of each marker was tested by using quantitated DNA of the appropriate Brucella species diluted in 10-fold serial dilutions. Each were tested in triplicate to determine the lower limit of detection (LLOD), defined as all three replicates displaying a positive growth curve and a reliable dissociation curve.
All primer sets used in the present assay were tested for specificity against a panel of near-neighbor organisms and human DNA as listed above. All organisms demonstrated no reactivity with the seven markers used when tested at the ~2-ng concentration used for testing Brucella spp. isolates (data not shown). The Bspp marker displayed no reactivity with any of the organisms. The LLOD for each marker was determined for detection (Bspp and Bsui) and, where applicable, reliable HRM curve analysis. These values ranged from 1 to 100 fg (Table (Table2).2). Four of the five melt curve dependent markers required only 100 fg for dependable curve interpretation (1 pg was needed for Bmel).
All Brucella isolates (n = 153, Table Table1),1), representing eight recognized species and multiple biovars, were tested by real-time PCR using seven primer sets: Bspp, Bmel, Bcan, Bmar, Bneo, Boa, and Bsui (Table (Table2).2). The Bspp marker specifically detects all members of the genus and displays only a positive amplification curve when Brucella species are present (data not shown). HRM curves for the six species-specific markers are illustrated in Fig. Fig.1.1. HRM analysis is required for Bmel, Bcan, Bmar, Bneo, and Boa markers (Fig. 1A to E) to determine the specific species of Brucella based upon unique dissociation profiles for each marker. B. melitensis isolates are shown in Fig. Fig.1A1A as possessing an earlier melting curve compared with all other isolates. This separation is due to a G→T transversion found within the int-hyp gene of B. melitensis species. A similar pattern is seen for B. canis isolates in Fig. Fig.1B1B when the Bcan primer was used; however, this is caused by a transition of G to A within the int-hyp locus. The marine strains (B. ceti and B. pinnipedialis) showed more variability in their melting profiles; however, they were easily distinguished from all other members when the Bmar primer was used (Fig. (Fig.1C).1C). These distinct HRM curves are attributed to species-specific changes within the targeted BP26 locus. These isolates demonstrated an overall higher melting curve temperature (shifted right) compared to the other six Brucella species. A transition (A→G) within the Glk gene accounts for the unique melting profile of B. neotomae when using the Bneo marker (Fig. (Fig.1D.)1D.) Lastly, both B. ovis and B. abortus display distinctive melting curves from one another and all other Brucella species when the Boa primer set was used (Fig. (Fig.1E).1E). The Boa primer set produces the earliest melting curve for B ovis strains due to two transitions (both G→A) within the targeted region of the Glk gene, whereas B. abortus contains a single transition (G→A) at a distinct locus within the same amplicon, thus resulting in a slightly higher melting temperature (Fig. (Fig.1E).1E). All other species display a dissociation curve that lies furthermost to the right due to the conservation of the three guanine residues at these two loci. The Bsui primer set displays a positive growth curve for B. suis strains only and fails to amplify all other species (Fig. (Fig.1F1F).
A recently described isolate (BO2) that has been proposed as a lineage of the new B. inopinata species was tested using the current assay (R. Tiller, unpublished data). A positive amplification using Bspp (data not shown) was followed by the six speciation markers. Although three markers (Bcan, Bmar, and Bneo) showed HRM curves that were consistent with known Brucella spp., the Bmel and Boa HRM profiles show unique dissociation curves that are different from any known Brucella isolates tested (Fig. (Fig.2).2). The Bmel marker shows a discernible curve shifted to the left of the B. melitensis isolates, attributed to a single transition of C→T (Fig. (Fig.2A),2A), while the HRM profile from the Boa assay displays a dramatic shift, again to the left, a finding indicative of a lower melting temperature due to three C→T transitions within the targeted amplicon (Fig. (Fig.2E).2E). The Bsui marker displayed no amplification, indicating a consistency with the isolate not being a B. suis species (Fig. (Fig.2F2F).
An earlier reported isolate (BO1) which prompted the proposal of B. inopinata as a novel species was also tested. This isolate did not display the same HRM curve patterns as BO2; however, it was unique because it did not match a profile for any of the known Brucella species. For each marker tested, this isolate always grouped with the “all other” Brucella species and was positive with Bspp and negative for Bsui.
We report the development of a real-time PCR assay that has taken advantage of the sensitivity of a postamplification high-resolution melt to differentiate members of the Brucella genus. By specifically targeting discriminating loci within the genomes of Brucella spp., this assay is able to generate distinctive melt curves under the same thermocycling program that are highly reproducible among different strains of the same species. This approach was used to accurately identify 152 of 153 Brucella isolates at the species level, demonstrating the reliability of the assay. Although the six-marker speciation panel proved to be sufficient for accurate determination of the species, we chose to also incorporate the Bspp marker. This marker serves as an initial identifying indicator of inclusion in the Brucella genus and detects all members of this genus. The absence of a positive amplification curve with this marker would preclude any further analysis of an isolate. All Brucelleae members tested demonstrated robust amplification curves, whereas near neighbors did not.
The HRM assay described here can identify novel or unusual isolates. Three reports describe real-time PCR assays for the detection of different Brucella spp.; however, none are based upon HRM analysis or are able to identify novel strains (6, 9, 10). Gopaul et al. recently reported a minor-groove binding-based assay to detect species-specific SNPs of the six classical members, as well as marine mammal strains (9). That study screened more than 300 isolates and found that B. suis bv5 was unable to be positively identified and that B. suis bv1 to bv4 could not be separated from B. canis isolates. Although the assay holds great utility, it requires the use of proprietary chemistry and two separate probes in each of the seven reaction wells which results in a substantial increase in cost. In contrast, the method described in the present study uses common and widely available reagents and equipment, along with inexpensive unlabeled oligonucleotides. To our knowledge, this assay is the first to specifically identify B. suis using real-time PCR. All other existing real-time PCR-based assays identify this species through indirect testing algorithms. Furthermore, this assay can identify unusual Brucella isolates such as BO1 and BO2. The unique melting profiles of these strains represent an important “plasticity” feature of this assay. By targeting multiple loci and employing HRM analysis, an atypical Brucella isolate can be recognized and be further examined.
This assay also proved successful for discriminating B. suis from B. canis, an objective that has proved to be challenging using real-time PCR approaches (7, 10). Interestingly, our assay was unable to accurately differentiate a B. suis bv4 from B. canis. This particular B. suis biovar has previously been reported to exhibit a genotypic pattern identical to B. canis, and it is still debated as to whether this is truly a unique biovar of B. suis (2, 11, 21). Further, B. canis has historically been shown to be very closely related to B. suis through a variety of molecular studies (21). This observation underscores the complexity involved with discriminating these two species of Brucella.
A main objective of our study was to provide a rapid molecular typing assay for Brucella isolates that is easily interpretable and possess the ability to identify unusual strains. Optimal CT values should be between 15 and 35 and display the characteristic sigmoid-shaped curve for reliable HRM data. Because sufficient quantity of DNA should be readily available from the isolates, this should not pose a problem. We observed reliable HRM curves down to 100 fg with all but one marker (Bmel), which required 1 pg. These differences may be due to the intrinsic amplification efficiencies within primer sets. Notwithstanding, these data demonstrate the sensitive nature of the assay and provide an easily achievable range of DNA concentrations for reliably performing the assay. This assay has not been tested on clinical specimens suspected of containing Brucella spp. However, the HRM technique has been used to detect other pathogens in clinical specimens and may be applicable with further optimization for such specimens (12, 14, 23).
The genetically monomorphic nature of the Brucella genus continues to be a challenge for both microbiologists and taxonomists. We present here a methodology that can enhance and supplement existing procedures to identify Brucella spp. This assay has the potential to improve the timely reporting of results, provide a means for greater characterization of isolates, aid in epidemiological investigations, and contribute to a more comprehensive typing system for this genus.
Published ahead of print on 6 January 2010.