Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-007-0262-z
CONCISE ARTICLE
Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis T. Skottman & H. Piiparinen & H. Hyytiäinen & V. Myllys & M. Skurnik & S. Nikkari
# Springer-Verlag 2007
Abstract This report describes the development of inhouse real-time PCR assays using minor groove binding probes for simultaneous detection of the Bacillus anthracis pag and cap genes, the Francisella tularensis 23 KDa gene, as well as the Yersinia pestis pla gene. The sensitivities of these assays were at least 1 fg, except for the assay targeting the Bacillus anthracis cap gene, which showed a sensitivity of 10 fg when total DNA was used as a template in a serial dilution. The clinical value of the Bacillus anthracis- and Francisella tularensis-specific assays was demonstrated by successful amplification of DNA from cases of cow anthrax and hare tularemia, respectively. No cross-reactivity between these species-specific assays or with 39 other bacterial species was noted. These assays may provide a rapid tool for the simultaneous detection and identification of the three category A bacterial species listed
T. Skottman : H. Piiparinen : H. Hyytiäinen : S. Nikkari (*) BC-Defence and Environmental Health Unit, Centre of Military Medicine and Centre for Biological Threat Preparedness, Tukholmankatu 8A, 00290 Helsinki, Finland e-mail:
[email protected] V. Myllys Microbiology Unit, Department of Animal Diseases and Food Safety Research, Finnish Food Safety Authority, Mustialankatu 3, 00790 Helsinki, Finland M. Skurnik Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, HUSLAB, P.O. Box 21, 00014 Helsinki, Finland
as biological threats by the Centers for Disease Control and Prevention.
Introduction Bacillus anthracis, Francisella tularensis, and Yersinia pestis are the three bacterial species considered to pose the greatest risk to public safety due to their potential to cause high rates of morbidity and mortality in humans; therefore, they are listed as category A biological agents by the Centers for Disease Control and Prevention (CDC). Deliberate exposure to aerosolized B. anthracis spores and F. tularensis can lead to inhalational anthrax and tularemia, respectively. Furthermore, in contrast to anthrax and tularemia, pneumonic plague caused by Y. pestis may spread from person to person. Rapid and accurate assays for microbial identification are essential to ensure proper medical intervention in the case of suspected intentional release of these agents. In this article we describe realtime PCR assays we developed based on minor groove binding (MGB) probes for the simultaneous detection of B. anthracis, F. tularensis and Y. pestis in a 96-well plate format, to be used in a mobile laboratory.
Materials and methods The bacterial strains used in this study are listed in Table 1. Clinical samples of animal tissue from a cow with anthrax and a hare with tularemia were received from the National Veterinary and Food Research Institute, Helsinki and Oulu, Finland, respectively. The anthrax sample was a formalinfixed lymph node from the intestine of a 1.5-year-old cow that had died under uncertain conditions. Culture analysis
Eur J Clin Microbiol Infect Dis Table 1 Bacterial species used with real-time PCR assays for detection of B. anthracis, F. tularensis and Y. pestis
a (1) DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; (2) Skurnik Laboratory Strain Collection; (3) Myllys Laboratory Strain Collection; (4) Forsman Laboratory Strain Collection, FOI, Umeå, Sweden; (5) HUSLAB, Helsinki, Finland b Represents 17 serotypes
Species
Strain(s)
Sourcea
Acidobacterium capsulatum Agrobacterium tumefaciens Bacillus anthracis Bacillus anthracis Bacillus cereus Bacillus licheniformis Bacilus mycoides Bacillus thurigiensis Borrelia burgdorferi Burkholderia multivorans Campylobacter jejuni Clostridium difficile Clostridium perfringens Clostridium septicum Clostridium sordellii Deinococcus geothermalis Enterobacter cloacae Escherichia coli Flavobacterium aquatile Francisella philomiragia Francisella tularensis Francisella tularensis Fusobacterium necrophorum Microbacterium barkerii Moraxella catarrhalis Planctomyces maris Pseudomonas aeruginosa Salmonella typhimurium Staphylococcus aureus Verromicrobium spinosum Yersinia bercovieri Yersinia enterocolitica Yersinia enterocolitica Yersinia frederikseni Yersinia intermedia Yersinia kristenseni Yersinia mollareti Yersinia pestis Yersinia pestis Yersinia pseudotuberculosis Yersinia ruckeri
DSM 11244 C58C1/RP4 ATCC 4229 Sterne 7702 ELMI 21, ELMI 170, ELMI 349, ELMI 466 ELMI 325 ELMI 44 ELMI 123 DSM 4680 DSM 13243 E1 2702/1/04, E1 4966/1/04, E1 893/1/03 ETA/an TYYP 630 ETA/an 1111, ETA/an 1701, ETA/an 1702 ETA/an 1236 ETA/an 1277 DSM 11300 tks461 C600/pYET6 DSM 1132 DSM 7535 LVS (ATCC 29684) 10 clinical culture isolates ETA/an 1318, ETA/an 1658 DSM 20145 035E DSM 8797 IATS ATCC 13311 ATCC 25923 DSM 4136 3016/84, 127/84 1309/80 20373/79 38/83, 3317/84 9/85 119/84, 317/82 92/84, IP 22404 EV76-c KIM D1 48 different strainsb RS41
(1) (2) (3) (3) (3) (3) (3) (3) (1) (1) (3) (3) (3) (3) (3) (1) (2) (2) (1) (1) (4) (5) (3) (1) (2) (1) (2) (2) (2) (1) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2)
followed by biochemical testing from the lymph node as well as PCR analysis [1] revealed that the infecting organism was B. anthracis. The tularemia sample consisted of formalin-fixed paraffin-embedded tissue of a dead hare that had been referred to the reference laboratory for suspected tularemia infection. The presence of F. tularensis had been confirmed by indirect immunogenic fluorescent staining. DNA from the bacterial isolates was purified using the automated MagnaPure LC System (Roche, Basel, Switzerland) and the LC DNA Isolation Kit III (Roche) according to the manufacturer’s protocols. The control strains of B. anthracis (Sterne 7702, pXO1+/pXO2- and ATCC 4229, pXO1-/pXO2+), F. tularensis (LVS) and
Y. pestis (EV76-c) were purified with the QiaAmp DNA MiniKit (Qiagen, Hilden, Germany). DNA from the tissue samples was purified with the QiaAmp DNA MiniKit as suggested by the manufacturer. Using the Primer Express version 2.0 software (Applied Biosystems, Foster City, CA, USA), we designed primer/ probe combinations, which were based on sequences available from the public NCBI database. The fluorescent reporter dye at the 5′ end of the probe was 6-carboxyfluorescein (FAM), and a non-fluorescent quencher was at the 3′ end. Optimal primer and probe concentrations were determined based on results of experiments performed in a matrix format, as described in the ABI 7300 (Applied
Eur J Clin Microbiol Infect Dis
Biosystems) instrument manual. B. anthracis-specific primers (pagF, pagR, capF, capR) and probes (pagTM and capTM) were designed from the pag gene (accession no. M22589) and from the cap gene (accession no. M24150) of the virulence plasmids pXO1 and pXO2, respectively. Y. pestis-specific primers (plaF, plaR) were selected to target the pla gene (accession no. M27820) of virulence plasmid pCP1. The forward primer was adapted from Loïez et al. [2] and the reverse primer, plaR, and probe, plaTM, were designed specifically for this study. The F. tularensis primers and probe targeted the 23 kDa gene (accession no. Y08861). Both primers (23F and 23R) were modified from Versage et al. [3] while the TaqMan probe, 23 kDaTM, was designed for this study. The sequences of the primers and probes used are shown in Table 2. All real-time PCR assays were performed in a final volume of 25 μl, consisting of 12.5 μl 2× TaqMan Universal PCR master mix (Applied Biosystems) containing dNTPs, MgCl2, reaction buffer, AmpErase®uracil-Nglycosylase (UNG), ROX passive reference and AmpliTaq Gold®, the primers as described below, and 2.5 μl of the template. Following optimization experiments, the PCR primer concentrations for the B. anthracis assays and for the F. tularensis assay were as follows: 300 nM of forward primer, 900 nM of reverse primer and 250 nM of probe. The PCR reactions for Y. pestis contained 50 nM of forward primer, 300 nM of reverse primer and 250 nM of probe. In addition, 2.5 μl of internal positive control (IPC) mix and 0.5 μl of IPC synthetic DNA, which were included in the TaqMan Exogenous Internal Positive Control Reagents Kit (Applied Biosystems), were used per reaction. Every PCR run included No Amplification Controls (NAC), where template was substituted with IPC Blocker, and No Template Controls (NTC), where template was substituted with water. Both controls were used as six replicates. A Table 2 Primers and probes used with real-time PCR assays for the detection of B. anthracis, F. tularensis and Y. pestis
bp base pairs a Based on accession numbers M22589 (pag), M27820 (pla), M24150 (cap), Y08861 (23 kDa)
Name pag pagF pagR pagTM cap capF capR capTM pla plaF plaR plaTM 23 kDa 23F 23R 23TM
positive control plasmid containing all target sequences was constructed. The pagA, pla, capB and 23 kDa DNA fragments were PCR amplified with the proofreading Pfu polymerase (Stratagene, CA, USA) from bacterial genomic DNA with gene-specific primers (data available by request). These fragments were sequentially ligated into the pUC19 vector resulting in a 3,502-bp plasmid that was named pMILFI. Real-time PCR assays were performed with the ABI 7300 instrument using the following thermocycling parameters: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s each at 95°C, and 1 min at 60°C. The obtained fluorescence data were analyzed using Sequence Detection software, version 1.2.2 (Applied Biosystems).
Results and discussion As measured using serial dilutions of target DNA, the sensitivities of the developed B. anthracis pag-gene, capgene, Y. pestis pla-gene and F. tularensis 23 kDa-gene assays were at least 1 fg, 10 fg, 1 fg and 1 fg of total DNA, respectively. This corresponds to the ability to detect approximately one bacterial cell per assay. The overall 10fold better sensitivity of the pag-gene assay as compared to the cap-gene assay corresponds with the respective B. anthracis plasmid copy numbers [4]. When the developed control plasmid was used as a template, the assay sensitivities varied between 20 and 200 plasmid copies. One copy of each target DNA sequence was incorporated per plasmid. To assess the specificity of the developed PCR assays, clinical and environmental bacterial species were chosen to represent the overall diversity of the bacterial kingdom at possible sample collection sites. Bacteria closely related to
Sequence
Positiona
Fragment size (bp)
5′-CGGATAGCGGC GTTAATC 5′-CAAATGCTATTTTAAGGGCTTCTT TT 5′-TAGAAACG CTAAACCGGATAT
3400–3418 3484–3459 3431–3452
85
5′-TTGGGAACGTGTGGATGATTT 5′-TCAGGGCGGCAATTCATAAT 5′-TAGTAATCTAGCTCCAATTGT
1106–1126 1174–1155 1133–1153
69
5′-GAAAGGAGTGCGGGTAATAGGTT 5′-CCTGCAAGTCCAATATATGGCATA 5′-TAACCAGCGCTTTTC
816–838 878–855 840–854
63
5′-TGAGATGATAACAAGACAACAGGTAAC 5′-GGATGAGATCCTATACATGCAGTAGGA 5′-CCATTCATGTGAGAACTG
551–578 634–608 589–606
84
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B. anthracis, F. tularensis and Y. pestis were examined more thoroughly. Bacterial strains listed in Table 1 were included for specificity testing of all assays, except the 48 Y. pseudotuberculosis strains, which were only used as template in the Y. pestis pla gene-specific assay. All samples were analyzed as duplicates. No false-positive results or cross reactivity caused by non-specific hybridization was observed. In addition, the IPC, which was incorporated in all assays, did not reveal the presence of inhibitors in any of the PCR reactions. To evaluate the potential use of our assay for the study of clinical samples, we performed three independent DNA isolations from the clinical samples of cow and hare tissue. When these samples were analyzed in our assay, Ct values ranging from 16 to 20 and 21 to 22 were detected, thus showing presence of B. anthracis and F. tularensis DNA, respectively (data not shown). The low Ct values suggest high clinical sensitivity. Several previous studies have evaluated individual realtime PCR detection methodologies for these three bacterial species. Uniquely, our assay enables simultaneous detection of the three species in one PCR run in a 96-well plate format, thus shortening the time needed for analysis. A highthroughput multiplex PCR system based on fluorescent-bead hybridization for detection of four biothreat agents [5] and a multi-pathogen oligonucleotide microarray for differentiating B. anthracis from other bacterial species [6] were recently described. In our study the thermocycling parameters of the three species-specific assays are homogenous, allowing simultaneous detection of all three bacterial species during one PCR run. We preferred to take advantage of the 96-well plate format and perform the PCR reactions concurrently in separate tubes, as well as to rely on the automated real-time probe detection methodology. In our hands, broad-range bacterial PCR followed by hybridization on a diagnostic array of oligonucleotide probes targeting species-specific variable regions of otherwise conserved genes has proven useful in research laboratory conditions [7]. However, the aim of our current study was to develop methods that could be used for rapid detection and identification of B. anthracis, F. tularensis and Y. pestis in a mobile diagnostic laboratory setting. B. anthracis and other members of the Bacillus cereus group exhibit an extremely high degree of genomic homology [8], thus making differentiation of these species challenging. Real-time PCR identification of B. anthracis based on both virulence plasmids pXO1 and pXO2 has been utilized earlier [9, 10]. In our study we designed an assay for the detection of pag and cap genes using the ABI 7300 instrument and MGB probes. According to Hurtle et al. [11] a chromosomal target is needed in conjunction with pXO1 and pXO2 because plasmid transfer among the B. cereus group has been observed. Since nonpathogenic B. anthracis
strains lacking either one or both plasmids also exist [12], the presence of both plasmids should be demonstrated concurrently. However, these exigent reactions were not observed in our study. Our method for the specific detection of Y. pestis was based on the plasminogen activator gene due to its estimated high copy number of 186 per bacterium [13]. The usefulness of this gene for clinical real-time PCR diagnostics has been shown in earlier studies as well [2, 14]. Initially, four regions of F. tularensis were selected as targets for real-time PCR detection, namely fopA, 23 kDa, tul4 and the ISFtu2 element. The 23 kDa gene was selected after preliminary testing (data not shown) based on assay sensitivity and also due to the importance of the gene in intracellular growth in macrophages, which is a central event in tularemia [15]. The 23 kDa gene has been used earlier as a target for real-time PCR detection of F. tularensis [3]. However, it has not previously been subjected to real-time PCR detection using MGB probes. Detection and identification of these bacteria by cultivation is time consuming and may pose a potential health risk for personnel, particularly when working in field conditions in the context of a mobile laboratory. In addition to rapidity, one of the benefits of molecular analysis is that the sample may be inactivated before analysis. Furthermore, the use of real-time PCR decreases the risk of pre-PCR contamination of samples with post-PCR amplicons—one of the greatest disadvantages of classical PCR. Our simultaneous assays achieved results in approximately 100 min from the beginning of analysis, excluding the time needed for sample processing. In conclusion, our MGB probe-based real-time PCR methodology can be used for the simultaneous identification of all three CDC category A bacterial species, namely B. anthracis, F. tularensis and Y. pestis. Rapid detection and identification of these agents will allow timely postexposure prophylactic treatment with antibiotics and/or vaccines before the onset of severe illness. However, results obtained with these assays in field conditions should be confirmed by other methodologies in reference laboratories. In the future, the 96-well plate format will enable future automation and high-throughput analysis of other selected agents. Acknowledgements This study was supported by a grant from the Finnish Scientific Advisory Board for Defence. We would like to thank Professor M. Vaara (HUSLAB, Helsinki, Finland) and Dr. M. Forsman (FOI, Swedish Defence Research Agency, Umeå, Sweden) for providing clinical cultures of F. tularensis and the F. tularensis LVS strain, respectively. Dr. M. Salminen (National Public Health Institute, Helsinki) is acknowledged for providing the laboratory facilities used for these studies. We also thank H. Hemmilä for excellent technical assistance and Dr. C. Heckman, as well as Ms. E. Nevalainen for editorial assistance. These experiments comply with the current laws of Finland.
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