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Technical Briefs |
1 Department of Medicine, Division of Infectious Disease, New Jersey Medical School, The University of Medicine and Dentistry of New Jersey, Newark, NJ;2 Department of Molecular Genetics, The Public Health Research Institute, Newark, NJ
aaddress correspondence to this author at: Division of Infectious Disease, New Jersey Medical School, 185 South Orange Ave., MSB A920C, Newark, NJ 07103; fax 973-972-0713, e-mail allandda{at}umdnj.edu
The advent of bioterrorism has highlighted the need for rapid, simple, and robust diagnostic assays to detect select agents. Mortality from select agents may be greatly reduced by prompt treatment (1); however, treatment may be delayed if diagnostic assays are outsourced to reference laboratories. Most bacterial species that would likely be used as bioterrorism agents infect the blood stream during the course of life-threatening disease. Furthermore, even "nonseptic" syndromes may produce hematogenous bacterial DNA that could be detected by a sensitive assay (2). This means that a rapid "molecular" version of a blood culture would fulfill many of the rapid diagnostic needs for biodefense.
Bacteria can be detected in blood and other sterile body sites by the identification of species-specific DNA sequences in their 16S rRNA genes. These species-specific sequences are flanked by conserved sequences, permitting most rRNA targets to be amplified by PCR using a limited set of "universal" primers (3). Real-time PCR is well suited for sensitive and specific pathogen detection because it is performed in hermetically sealed wells, which greatly reduces the risk of cross-contamination, and it does not require post-PCR analysis (4). Real-time PCR assays have been developed for some select agents, most of which use fluorogenic 5'-nuclease (TaqMan) probes (5)(6)(7). However, TaqMan probes are difficult to use in multiplex PCR assays (8)(9). In contrast, molecular beacons are real-time PCR probes that are particularly amenable to multiplexing (10). They can be labeled with differently colored fluorophores (11), use a common nonfluorescent quenching moiety (9), and have thermodynamic properties that favor highly specific detection of nucleic acid sequences (12).
Here we describe a real-time PCR assay that simultaneously detects four bacterial agents that could be used in bioterrorism. This assay is specifically designed to test sterile body fluids, where a rapid and simple assay would be beneficial. We developed a flexible assay format that can easily be adapted to the wide range of spectrofluorometric thermal cyclers that are in common use, including thermal cyclers that have only one- or two-color capabilities, and others that can detect four or more colors simultaneously.
Phenol–chloroform extraction of DNA from standard strains of Bacillus anthracis Vollum and Sterne, Yersinia pestis CO92, and Burkholderia mallei (ATCC 23344) was performed as described previously (13) in a biosafety level III laboratory certified to work with select agents (registration number 20011016-798; entity number C20031123-0125). A 180-bp amplicon for use as template in Francisella tularensis assays was also constructed in vitro from two overlapping oligonucleotides (Invitrogen). DNA was also extracted from clinical isolates of Staphylococcus aureus, S. epidermidis, Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, and Serratia marcescens to serve as controls.
The conservation of 16S rRNA gene sequences among bacteria enabled us to design primers FUHP (5'-GTGGACTTAGATACCCTGGTAGTCCAC-3'; underlined sequence indicates additional nucleotides added to create a hairpin structure) and RUP (5'-GCGTTGCATCGAATTAA-3') to amplify short segments of the 16S rRNA genes of Y. pestis, F. tularensis, and B. mallei by PCR. The benefits of hairpin-shaped primers have been noted previously (14)(15)(16). A second primer pair, FBa (5'-TGACGACAACCATGCACC-3') and RBa (5'-ATGTGGTTTAATTCGAAGCAA-3'), was designed to amplify a segment of the 16S rRNA gene of B. anthracis.
We designed molecular beacons (Table 1
) that bound to amplicons generated from F. tularensis, B. mallei, and B. anthracis and that could not bind to amplicons generated by other significant human pathogens. However, the Y. pestis molecular beacon also bound to amplicons generated from K. pneumoniae, E. coli, E. cloacae, and S. marcescens, which share an identical 16S rRNA gene target sequence with Y. pestis. To standardize assay operating conditions, we also adjusted the probe and arm sequences of each molecular beacon so that all had similar melting temperatures in the presence of perfectly complementary targets.
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We tested the ability of each molecular beacon to specifically identify its target in a single-color assay using multiple wells of a 384-well assay plate. The molecular beacons designed to detect Y. pestis (Table 1
, probe 1), F. tularensis (probe 2), B. mallei (probe 3), and B. anthracis (probe 4) were labeled with the fluorophore fluorescein for these experiments. A 384-well assay plate was prepared containing a series of identical four-well assays with four different probes (200 nM) in each of the four wells. In addition, the wells contained 1x PCR buffer (Applied Biosystems); 4 mM MgCl2; 250 µM each of dATP, dCTP, dGTP, and dTTP; 0.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems); 2.5 pmol each of both sets of primers; and 1 µL of template DNA in a final volume of 5 µL. Real-time PCR was performed with a 7900HT Prism spectrofluorometric thermal cycler (Applied Biosystems). The reaction mixtures were incubated for 10 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 55 °C for 60 s, and 72 °C for 30 s. Fluorescence was measured in every well or tube during each annealing step throughout the course of each reaction. The "threshold cycle" was automatically determined by the computer program controlling the spectrofluorometric thermal cycler. Significant fluorescence appeared only in the wells containing a molecular beacon complementary to the target sequence of the bacterial DNA added to that well.
We also studied whether the assay could be multiplexed into a single-color, single-well screening assay to identify the presence of a select agent in a simplified format but not to distinguish among them. All four fluorescein-labeled molecular beacons and both primer pairs were combined in a single reagent mixture. DNA from one of the select agents or control organisms was added to each well, and real-time PCR was performed. As expected, a detectable fluorescence signal developed in every well that contained DNA complementary to one of the four molecular beacons present in the well (data not shown). In both these assay formats, no fluorescence signals appeared in any well when DNA from S. aureus, S. epidermidis, S. pneumoniae, B. cereus, or a no-DNA control was added. Wells containing probe 1 fluoresced in the presence of Y. pestis, K. pneumoniae, E. coli, E. cloacae, and S. marcescens (all of which share the same molecular beacon target sequence).
We also estimated the lower limit of detection of the single-color multiplex assay. Triplicate DNA samples from each select agent were serially diluted in water and tested using the above protocol. We found positive PCR signals in all three replicates containing DNA extracted from the equivalent of 
50 bacilli for B. mallei and 20 bacilli for the other bacteria.
The optimum biodefense assay should be able to both detect and distinguish among all select agents in a single assay well or tube. Each molecular beacon in the multiplex assay would have to be individually distinguishable to accomplish this goal. We met these design criteria by labeling each molecular beacon with a different fluorophore and performed the assay in a SmartCycler II (Cepheid) that could independently monitor the fluorescence generated by each fluorophore in an assay tube containing all four fluorophores. We selected each of the four fluorophores so that their emission maxima were well spaced from each other across the visible spectrum. All four molecular beacons and both primer pairs were multiplexed into a single reagent mixture to a final volume of 25 µL. DNA from one of the select agents or control organisms was added to each well, and real-time PCR was performed. A detectable fluorescence signal of the appropriate color developed in every well that contained DNA complementary to one of the four molecular beacons present in the well (Fig. 1
). Each experiment was repeated at least three times to test its reproducibility.
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The importance of bacterial diagnostics in sterile body fluids is supported by current medical practice in which blood cultures are performed on virtually all ill patients with fevers and a suspected bacterial source (17). The ability of molecular beacons to be labeled with differently colored fluorophores, and the development of instruments that are able to detect them, raises the possibility that highly multiplexed PCR assays can be designed to serve as "molecular blood cultures", replacing current culture-based techniques. The availability of PCR screening assays would greatly advance our ability to rapidly detect a broad range of infections, including those introduced by bioterrorism. Although the Y. pestis-specific molecular beacon was also able to hybridize to other Enterobacteriaceae, we do not view this as a disadvantage. This feature expands the range of the assay, enabling the detection of other common agents that cause sepsis. Importantly, the assay did not detect pathogens from skin flora that can occasionally contaminate a blood draw (in contrast, the presence of Enterobacteriaceae in a sterile body site is almost always indicative of disease). Future assays can be designed to specifically detect Y. pestis by including a third primer pair and a molecular beacon that differentiates between Y. pestis and other Enterobacteriaceae.
Acknowledgments
This work was supported by Public Health Service Grant AI-056689 from the National Institutes of Health and by Department of Defense Grant DAMD 17-01-1-0787 from the United States Army Medical Research Materiel Command. Dr. Varma-Basil received support in the form of an overseas Associateship from the Indian Government, Department of Biotechnology. David Alland, Fred Kramer, and Salvatore Marras are among a group of co-inventors who hold patents in molecular beacons and receive income from licensing agreements.
Footnotes
1 current affiliation: Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India 
References
The following articles in journals at HighWire Press have cited this article:
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  S. H. Thomas, E. Padilla-Crespo, P. M. Jardine, R. A. Sanford, and F. E. Loffler Diversity and Distribution of Anaeromyxobacter Strains in a Uranium-Contaminated Subsurface Environment with a Nonuniform Groundwater Flow Appl. Envir. Microbiol., June 1, 2009; 75(11): 3679 - 3687. [Abstract] [Full Text] [PDF]
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 H. H. El-Hajj, S. A. E. Marras, S. Tyagi, E. Shashkina, M. Kamboj, T. E. Kiehn, M. S. Glickman, F. R. Kramer, and D. Alland Use of Sloppy Molecular Beacon Probes for Identification of Mycobacterial Species J. Clin. Microbiol., April 1, 2009; 47(4): 1190 - 1198. [Abstract] [Full Text] [PDF]
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 E. S. Gabitzsch, R. Vera-Tudela, R. J. Eisen, S. W. Bearden, K. L. Gage, and N. S. Zeidner Development of a Real-time Quantitative PCR Assay to Enumerate Yersinia pestis in Fleas Am J Trop Med Hyg, July 1, 2008; 79(1): 99 - 101. [Abstract] [Full Text] [PDF]
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 J. J. Li, Y. Chu, B. Y.-H. Lee, and X. S. Xie Enzymatic signal amplification of molecular beacons for sensitive DNA detection Nucleic Acids Res., April 1, 2008; 36(6): e36 - e36. [Abstract] [Full Text] [PDF]
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P. E. Saikaly, M. A. Barlaz, and F. L. de los Reyes III Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate Appl. Envir. Microbiol., October 15, 2007; 73(20): 6557 - 6565. [Abstract] [Full Text] [PDF]
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M. R. Pingle, K. Granger, P. Feinberg, R. Shatsky, B. Sterling, M. Rundell, E. Spitzer, D. Larone, L. Golightly, and F. Barany Multiplexed Identification of Blood-Borne Bacterial Pathogens by Use of a Novel 16S rRNA Gene PCR-Ligase Detection Reaction-Capillary Electrophoresis Assay J. Clin. Microbiol., June 1, 2007; 45(6): 1927 - 1935. [Abstract] [Full Text] [PDF]
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 K. Tomioka, M. Peredelchuk, X. Zhu, R. Arena, D. Volokhov, A. Selvapandiyan, K. Stabler, J. Mellquist-Riemenschneider, V. Chizhikov, G. Kaplan, et al. A Multiplex Polymerase Chain Reaction Microarray Assay to Detect Bioterror Pathogens in Blood J. Mol. Diagn., October 1, 2005; 7(4): 486 - 494. [Abstract] [Full Text] [PDF]
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