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Journal of Clinical Microbiology, November 2005, p. 5771-5774, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5771-5774.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of a Real-Time PCR TaqMan Assay for Rapid Identification and Differentiation of Burkholderia pseudomallei and Burkholderia mallei

Jana M. U'Ren,1 Matthew N. Van Ert,1 James M. Schupp,1 W. Ryan Easterday,1 Tatum S. Simonson,1 Richard T. Okinaka,2 Talima Pearson,1 and Paul Keim1*

Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 86011-5640,1 Los Alamos National Laboratory, Los Alamos, New Mexico 875452

Received 8 June 2005/ Returned for modification 19 July 2005/ Accepted 24 August 2005


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ABSTRACT
 
A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia.


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TEXT
 
The intracellular, pathogenic bacteria Burkholderia pseudomallei and Burkholderia mallei are the causative agents of melioidosis and glanders, respectively. Both pathogens are considered potential bioweapons and are listed as category B biothreat agents by the U.S. Centers for Disease Control and Prevention (16). Melioidosis is a serious infectious disease of humans and animals in the areas of endemicity in Southeast Asia and northern Australia. B. pseudomallei, a widely distributed environmental saprophyte, is resistant to a variety of adverse environmental conditions (3). Human and animal infection can occur through contact with the organism in the environment via ingestion or inhalation or through open wounds and skin abrasions (3). Melioidosis has a case fatality rate of 39.5%, and untreated septicemia is fatal in up to 80% of cases (9, 19). Glanders is a globally distributed disease of horses, mules, and donkeys (5) but has been mostly eradicated from North America by the destruction of large numbers of animals (4). Glanders can be contracted by humans through exposure to infected animals or aerosols (11), and it is difficult to distinguish from melioidosis due to their clinical and genetic similarities (10, 14).

Traditional methods for diagnosing melioidosis and glanders include serological and biochemical analyses of isolated cultures (18). Unfortunately, these methods may require up to 3 to 4 days to obtain the results, can be ambiguous, and are not amenable to high-throughput analysis (1, 18). More recently, rapid molecular techniques have become recognized as important tools for identification of B. pseudomallei and B. mallei (7), in order to allow more successful patient treatment, epidemiological tracking, and initial efforts to limit further dissemination (1, 8).

Current molecular methods for detecting these pathogens involve gene sequencing (16S and 23S rRNA genes) (2, 8), multiplex PCR (13), and plus-minus real-time PCR of the type III secretion system genes (17). These studies have shown discrimination between B. pseudomallei and B. mallei, but sequencing and endpoint multiplex PCR impose practical limits on throughput. The plus-minus real-time PCR of the type III secretion system gene assay has higher throughput but requires three separate PCRs in order to correctly identify B. pseudomallei, B. mallei, and B. thailandensis. In contrast, we present a real-time PCR allelic-discrimination assay designed around an informative single-nucleotide polymorphism (SNP) that can be used to discriminate between these bacterial species in a single reaction.

In order to identify informative SNPs that distinguish between B. pseudomallei and B. mallei, we examined multilocus sequence-typing data from 46 diverse B. pseudomallei, 10 B. mallei, and 9 B. thailandensis isolates (R. Okinaka, unpublished data) (Fig. 1). A synonymous SNP in a putative antibiotic resistance gene, termed P27, at base 2351851 of chromosome 1 (B. pseudomallei strain K96243; GenBank accession no. NC_006350) fit the criterion of differentiating B. pseudomallei from B. mallei.



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  		FIG. 1. Sequence alignment of the P27 gene fragments from B. pseudomallei and near neighbors. The rows indicate the sequences of (A) the primers and probes used in the assay; (B) 42 B. pseudomallei strains from Bangladesh, Ecuador, France, The Netherlands, Indonesia, Italy, Kenya, Madagascar, Malaysia, Philippines, Singapore, Sweden, Thailand, United Kingdom, Vietnam, and Australia; (C) 2 B. pseudomallei strains from Australia; (D) 2 B. pseudomallei strains from Malaysia and Thailand; (E) 10 B. mallei strains from China, Hungary, India, and Turkey; (F) 9 B. thailandensis strains from Thailand; (G) B. cepacia GenBank strain R18194; and (H) B. cepacia GenBank strain R1808. Light shading indicates primer and probe locations. Dark shading indicates nucleotide differences between near neighbors and the majority of B. pseudomallei isolates. The asterisk indicates the position of the synonymous SNP; + indicates degeneracy in the forward primer.

Based upon these sequences, we designed a TaqMan-minor-groove binding (MGB) allelic-discrimination assay around the synonymous SNP using Primer Express software (Applied Biosystems, Foster City, CA). One probe was designed to specifically hybridize to the B. pseudomallei sequence (5'-6FAM-CGCTCGAGGTTGA-3'-MGB), and the other was designed to hybridize to the B. mallei sequence (5'-VIC-CGCTCGAGATTGA-3'-MGB). Forward and reverse primers were positioned over mutations in the B. thailandensis and B. cepacia sequences to prevent amplification of these species (Fig. 1). Since we observed a single-nucleotide difference in the priming site between some isolates of B. pseudomallei and all B. mallei isolates, we incorporated degeneracy in the forward primer to accommodate amplification (Fig. 1).

Real-time PCR was conducted in a 10.0-µl reaction mixture containing 600 nM of both forward (5'-GCGTAGTCGCCCYTGAAGACT-3') and reverse (5'-GCCGAGGCGCAGTATCA-3') primers, 250 nM of each probe, 1x Applied Biosystems Master Mix, and 1.0 µl of template. Thermal cycling was performed on an ABI 7900 HT sequence detection system (Applied Biosystems) under the following conditions: 50°C for 2 min, 95°C for 10 min, and 50 cycles of 95°C for 15 s and 60°C for 1 min.

To evaluate this SNP locus as a species-specific marker for B. pseudomallei and B. mallei, the assay described above was used to genotype a collection of 302 B. pseudomallei and 37 B. mallei human, animal, and environmental isolates from a broad geographic and genetic range (J. Schupp, unpublished data) (Table 1). Species identification was confirmed by bacterial culture and/or biochemical tests in the laboratory of origin. DNA was isolated by either a phenol-chloroform or crude heat lysis method. Of the 302 B. pseudomallei isolates screened, all were shown to have the G allele at P27; this is consistent with our sequence data from a subset of 46 B. pseudomallei isolates (Fig. 1). All 37 B. mallei strains in the study amplified and exhibited the alternate P27 genotype (A allele), which agrees with sequence data from a subset of 10 B. mallei isolates (Fig. 1). To test the assay for cross-reactivity, a panel of near neighbors was selected after a microbial-genome GenBank BLAST search identified B. pseudomallei, B. mallei, B. thailandensis, and B. cepacia as the only significant matches. The near-neighbor panel screened included seven B. thailandensis, five B. cepacia, and two B. gladioli isolates and two Ralstonia spp. As expected, all 16 near neighbors failed to amplify due to the nucleotide mismatches in the primer and probe hybridization sites (Table 1 and Fig. 2).


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  		TABLE 1. B. pseudomallei, B. mallei, and genetic near-neighbor isolates used in the study



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  		FIG. 2. Data points (blue) along the y axis show 26 B. pseudomallei isolates. Data points (red) along the x axis show 18 B. mallei isolates. Data points (black) in the bottom left corner show four negative template controls (NTC) and 10 near neighbors, including 5 B. cepacia isolates, 2 B. gladioli isolates, 2 Ralstonia spp., and 1 B. thailandensis isolate. All DNAs were normalized at 100 pg/µl. The average cycle threshold values were as follows: B. pseudomallei, 30.41, and B. mallei, 26.69. There was no amplification for the genetic near neighbors and negative template controls.

To test the detection limit of the assay, a subset of DNAs from each species was quantified using a Pico Green quantification kit (Molecular Probes, Eugene, OR) and a minifluorometer (Turner Biosystems, Sunnyvale, CA). A dilution series with template levels ranging from 1 ng to 10 fg was generated from three quantified DNAs for each species and used in the TaqMan assay. The assay consistently detected and genotyped B. pseudomallei and B. mallei DNA template concentrations as low as 100 fg, which was calculated to be ~27 genome equivalents. Sporadic amplification at 10 fg was observed (data not shown).

Specific and sensitive diagnostic assays for the identification of pathogenic bacteria are critical for clinical and biodefense-related applications (7). The pathogenicity of B. pseudomallei and B. mallei and their potential use as biowarfare/bioterrorism agents underscore the importance of rapid detection and identification of these pathogens. Recently, SNPs have been shown to be important markers for the identification of biothreat agents (6). This is due to the fact that SNPs are considered to be evolutionarily stable markers because they are less likely than other markers to mutate to either a novel or ancestral state (12, 15). Thus, they are powerful phylogenetic markers for the molecular typing of bacteria. Our results suggest that the P27 SNP is a reliable way to identify and differentiate B. pseudomallei and B. mallei (Fig. 2). This conclusion is supported by the presence of species-specific alleles in over 300 B. pseudomallei and 37 B. mallei isolates from diverse geographic locales.

While any assay would benefit from further validation against new isolates, the present assay has been extensively tested against genetically and geographically diverse strains. Whereas additional changes within the gene in B. pseudomallei and B. mallei could potentially cause false-negative results in the assay, this was not observed in our highly diverse set of samples. Similarly, possible false positives could result from an unknown near-neighbor strain with sequence similarity to B. pseudomallei and B. mallei. However, the second scenario is highly unlikely considering the lack of significant GenBank BLAST results in other bacterial species. In addition, the 16 near-neighbor isolates that were screened contained extensive sequence variation in the probe and primer regions, which prevented amplification and/or detection (Fig. 1).

In conclusion, our results indicate that the P27 SNP has the ability to differentiate B. pseudomallei and B. mallei in a globally and genetically diverse panel of isolates. This real-time assay represents a potentially valuable diagnostic tool for detecting and identifying these species, since it is considerably more rapid and specific than current molecular or clinical methods (3, 19). Further studies will test the assay against additional diverse isolates and optimize the assay for specific low-level detection of B. pseudomallei and B. mallei in clinical and environmental samples.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the Department of Homeland Security.

We thank Bart Currie (Menzies School of Health Research, Darwin, Northern Territory, Australia), David DeShazer (USAMRIID, Fort Detrick, Maryland 21702-5011), Jay Gee (Centers for Disease Control and Prevention, Atlanta, Georgia 30333), Steven Harvey (U.S. Army Edgewood Chemical and Biological Center, Aberdeen Proving Ground, Maryland), Richard Robison (Brigham Young University, Provo, Utah 84602), Nicholas J. White (Wellcome Unit, Mahidol University, Bangkok, Thailand), Tyrone Pitt (Health Protection Agency, London, United Kingdom), David Dance (Plymouth Department of Microbiology, Derriford Hospital, United Kingdom), and Rasana Wongratanacheewin (Khon Kaen University, Khon Kaen 40002, Thailand) for providing the DNAs used in this study.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640. Phone: (928) 523-1078. Fax: (928) 523-0639. E-mail: Paul.Keim{at}nau.edu. Back


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Journal of Clinical Microbiology, November 2005, p. 5771-5774, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5771-5774.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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