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A simple method for the rapid removal of Bacillus anthracis spores from DNA preparations
Journal of Microbiological Methods 76 (2009) 212–214
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Note
A simple method for the rapid removal of Bacillus anthracis spores from DNA preparations ☆ Leslie A. Dauphin ⁎, Michael D. Bowen Bioterrorism Rapid Response and Advanced Technology (BRRAT) Laboratory, Division of Bioterrorism Preparedness and Response (DBPR), National Center for Preparedness, Detection, and Control of Infectious Diseases (NCPDCID), Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA
a r t i c l e
i n f o
Article history: Received 8 October 2008 Accepted 9 October 2008 Available online 28 October 2008 Keywords: Bacillus anthracis Bacillus anthracis spores Anthrax Bioterrorism
a b s t r a c t This study establishes a filtration method for the safe removal of Bacillus anthracis spores which may contaminate DNA preparations. Centrifugal filtration with 0.1-μm filter units can be used following extraction of DNA from B. anthracis spores to render samples safe without compromising the sensitivity of diagnostic real-time PCR assays for B. anthracis. Published by Elsevier B.V.
Bacillus anthracis, the causative agent of anthrax, is regarded as one of the most effective biological weapons because of the potential for large-scale dissemination of spores (Rotz et al., 2002). In October 2001, the Ames strain of B. anthracis was used as a weapon of bioterrorism in the United States (Hoffmaster et al., 2002a; Jernigan et al., 2001). Since the 2001 anthrax attack, many nucleic acid amplification based assays for the detection of B. anthracis in clinical and environmental samples have been developed (Bell et al., 2002; Ellerbrok et al., 2002; Hoffmaster et al., 2002b; Ryu et al., 2003). The 2001 anthrax attack demonstrated that diagnostic laboratories are likely to receive large numbers of environmental specimens during a bioterrorism investigation (Heller et al., 2002; Kiratisin et al., 2002; Luna et al., 2003). Environmental specimens may contain spores, and it has been reported that most commercial extraction kits do not completely deplete B. anthracis spores from samples (Dauphin et al., 2008a; Panning et al., 2007). Studies have used centrifugal filter units during processing procedures for B. anthracis (Marston et al., 2005; Sue et al., 2007); however, none have established the optimal filter types and conditions for the elimination of spores from samples. The purpose of this study was to establish a rapid method for the complete removal of B. anthracis spores from DNA samples so that subsequent PCR testing can be carried out safely at lower levels of biocontainment. ☆ Disclaimer Statement: "This article reflects the findings and conclusions of the conducted research. Names of vendors or manufacturers are provided as examples of available product sources; inclusion does not imply endorsement of the vendors, manufacturers or products by the Centers for Disease Control and Prevention or the U.S. Department of Health and Human Services." ⁎ Corresponding author. BRRAT Laboratory, DBPR, NCPDCID, CDC, Mail Stop G-42, 1600 Clifton Road, Atlanta, GA 30333, USA. Tel.: +1 404 639 4991; fax: +1 404 639 4234. E-mail address: [email protected] (L.A. Dauphin). 0167-7012/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.mimet.2008.10.009
All procedures using virulent B. anthracis spores were performed in a biosafety level 3 laboratory. Eight strains of B. anthracis were used: K1340, K1694, K2478, K2484, K2762, K4596, K5135, and K9002. Spore suspensions were prepared using methods which have been described (Dauphin et al., 2008b). The Ultrafree-MC filter units in 1.5-ml tubes with microporous membranes and pore sizes of 0.1 μm, 0.22 μm, and 0.45 μm (Millipore Corporation, Billerica, MA) were evaluated for their ability to remove viable spores from samples as measured by performing colony counts of filtered sample extracts. A total of 100 filter units were tested for each pore size evaluated. A 100-μl aliquot of strain K1694 (Ames) spores at a concentration of 106 CFU/ml in phosphate-buffered saline (PBS) (0.01 M, pH 7.4) was loaded in each filter unit with great care so as to not puncture the filter membranes, and spore suspensions were centrifuged at 12,000 ×g for 2 min as recommended by the manufacturer. Immediately following filtration, the entire volume of each filtrate was spread onto Trypticase soy agar with 5% (vol/vol) sheep blood plates (TSAB) (BD Diagnostic Systems, Sparks, MD) and incubated for up to 48 h at 37 °C. Four commercial extraction kits were used: the ChargeSwitch gDNA Mini Bacteria Kit (Invitrogen, Carlsbad, CA), NucliSens Isolation Kit (BioMerieux, Inc., Durham, NC), Puregene Genomic DNA Purification Kit (Qiagen Inc., Valencia, CA), and QIAamp DNA Blood Mini Kit (Qiagen Inc., Valencia, CA). The kits were selected because it has been shown that sample extracts prepared from B. anthracis spores (≥105 CFU/ml) using these kits may be contaminated with viable spores (Dauphin et al., 2008a). A total of one hundred ninety-two samples were processed using the four kits, and the sample extracts were filtrated as described above. A 3-target (plasmid pXO1, plasmid pXO2, chromosome) real-time PCR assay (Hoffmaster et al., 2002b) was used to determine the effects
L.A. Dauphin, M.D. Bowen / Journal of Microbiological Methods 76 (2009) 212–214 Table 1 Comparison of centrifugal filter units for the removal of spores from B. anthracis spore suspensions Filter pore size (μm)a
Viable spores in filtrateb No. pos/no. tested
0.1 0.22 0.45
Confluent growth
b10 colonies
Efficiency (%)c
1/100 1/100 1/100
0/100 2/100 7/100
99 97 92
a Centrifugal filter units were evaluated using 100-μl aliquots of virulent B. anthracis Ames strain spores at a concentration of 106 CFU/ml, which were centrifuged at 12,000 ×g for 2 min. b The entire volume of the filtrates was plated onto Trypticase soy agar with 5% (vol/vol) sheep blood plates and viability was determined by direct observation of plates for colonies. Two different growth densities were observed on TSAB plates; confluency, and b 10 colonies. c The efficiency of the filter units was calculated by dividing the number of samples which contained no viable spores as measured by colony counts of filtered sample extracts, divided by the number of samples tested, multiplied by 100.
213
attributable to the centrifugation conditions. Since centrifugation at 6000 to 10,000 ×g for 2 min resulted in 100% removal of spores, these conditions were selected. To determine whether the filtration method affected the level of detection for B. anthracis DNA, real-time PCR results for filtered versus unfiltered DNA extracts were compared (Fig. 1). The filtration method did not significantly affect the detection levels for any of the three real-time PCR targets when compared by paired t test (two-tailed). The differences in mean CT values for the chromosomal target, 25.1 for filtered samples versus 24.7 for unfiltered samples, were not found to be significant (P = 0.53; n = 18, Fig. 1A), as were the differences in the mean CT values for the pXO1 target (24.1 and 23.6, respectively,
of the filtration method on the detection levels of B. anthracis DNA. Reactions were performed in 25-μl volumes including 5 μl of filtered or unfiltered sample extract using reagent concentrations, cycling conditions, and instrumentation as described previously (Dauphin et al., 2008b). Table 1 shows the results for viability testing of filtrates from B. anthracis Ames strain spores. Overall, filter units with a pore size of 0.1 μm were the most efficient at removing spores from samples. The efficiency of the 0.1-μm filter units was 99%, whereas the 0.22-μm and 0.45-μm filter units had efficiencies of 97% and 92%, respectively. Two different growth densities were observed on TSAB plates; confluency, and a few (b10) colonies. For each of the filter unit sizes, one sample filtrate out of 100 produced confluent growth on the TSAB plate suggesting that the centrifugation conditions resulted in a 1% failure rate for each pore size evaluated. In addition, two 0.22-μm filter unit filtrates, and seven 0.45-μm filter unit filtrates, produced growth of b10 colonies on TSAB plates indicating that for these samples, a few spores passed through the filter units in contrast to the confluent growth observed in cases of complete filter failure. To establish conditions which provide 100% removal of viable spores from samples, we further evaluated the 0.1-μm filter units. Table 2 shows the efficiency of 0.1-μm filter units under a range of centrifugation conditions. Centrifugation at 12,000 ×g for 1 to 2 min resulted in 99% efficiency, (1% failure rate characterized by confluent growth in culture) whereas centrifugation at 6000 to 10,000 ×g for 2 min consistently provided 100% removal of spores. These results suggest that the observed 1% failure rate for the 0.1-μm filter units was Table 2 Efficiency of 0.1-μm filter units for the removal of spores from B. anthracis spore suspensions under varying centrifugation conditions Conditionsa
Viable spores in filtrateb
Time (min)
Force (×g)
No. pos/no. tested
Efficiency (%)c
2.0 1.5 1.0 0.5 2.0 2.0 2.0
12,000 12,000 12,000 12,000 10,000 8000 6000
1/100 1/100 1/100 0/100 0/100 0/100 0/100
99 99 99 100 100 100 100
a
Centrifugation conditions were evaluated using 100-μl aliquots of live B. anthracis Ames strain spores at a concentration of 106 CFU/ml. b The entire volume of the filtrates was plated onto Trypticase soy agar with 5% (vol/vol) sheep blood plates and viability was determined by direct observation of plates for colonies. All filtrates which were positive for viable spores resulted in confluent growth in culture. c The efficiency for the removal of viable spores was calculated by dividing the number of samples which contained no viable spores as measured by colony counts of filtered sample extracts, divided by the number of samples tested, multiplied by 100.
Fig. 1. Results of TaqMan real-time PCR analysis of filtered versus unfiltered sample extracts. DNA was prepared from virulent B. anthracis Ames strain spores at a concentration of 106 CFU/ml using the QIAamp kit and assayed using the (A), BA3 chromosome-specific primer/probe set; (B), BA2 pXO1 plasmid-specific primer/probe set; and (C), BA1 pXO2 plasmid-specific primer/probe set. The results were plotted as the mean relative fluorescence for filtered and unfiltered sample extracts, and no template controls (NTC) versus the cycle numbers. The differences in CT values between filtered versus unfiltered samples were not found to be significant using a paired t test (two tailed) for the chromosome (P = 0.53; n = 18), pXO1 plasmid (P = 0.52; n = 18) and pXO2 plasmid (P = 0.46; n = 18) targets.
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P = 0.52; n = 18, Fig. 1B) and the pXO2 target (24.7 and 23.8, respectively, P = 0.46; n = 18, Fig. 1C). Further, the method was tested on triplicate DNA samples prepared from the spores of eight strains of B. anthracis using four commercial extraction kits and the results indicated 100% removal of viable spores in 0.1 volume test aliquots. This study established a filtration method for the rapid removal of B. anthracis spores from DNA preparations without significantly affecting the detection of B. anthracis DNA in real-time PCR. Centrifugal filtration with 0.1-μm filter units at 6000 to 10,000 ×g for 2 min can be used by diagnostic laboratories involved in testing of environmental samples during investigations of suspected events of anthrax bioterrorism to rapidly remove spores from DNA samples prior to testing. Safety testing by culture is the only way to guarantee that DNA extracts are free of infectious spores but in situations when time-to-results is critical and the testing laboratory cannot wait 24 to 48 h for safety testing results, the method developed in this study will significantly reduce the frequency of spore contamination in DNA extracts. We thank John Ridderhof, Pamela Diaz, Harvey Holmes, Patricia Fields, Bruce Newton, Heather Stang, Justin Schrager, Rebecca Hutchins, and Kenyatta Stephens for their critical review of this manuscript. B. anthracis is a select agent and its possession, use, and transfer is regulated by the U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, and the U.S. Department of Agriculture, Animal and Plant Health Inspection Service. The select agent regulations have mandatory reporting requirements for identification of select agents in diagnostic specimens. References Bell, C.A., Uhl, J.R., Hadfield, T.L., David, J.C., Meyer, R.F., Smith, T.F., Cockerill III, F.R., 2002. Detection of Bacillus anthracis DNA by LightCycler PCR. J. Clin. Microbiol. 40, 2897–2902. Dauphin, L.A., Moser, B.D., Bowen, M.D., 2008a. Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus anthracis spores and comparison of DNA yields from spores and spiked environmental samples. J. Microbiol. Methods. doi:10.1016/j.mimet.2008.09.004. Dauphin, L.A., Newton, B.R., Rasmussen, M.V., Meyer, R.F., Bowen, M.D., 2008b. Gamma irradiation can be used to inactivate Bacillus anthracis spores without compromising the sensitivity of diagnostic assays. Appl. Environ. Microbiol. 74, 4427–4433.
Ellerbrok, H., Nattermann, H., Ozel, M., Beutin, L., Appel, B., Pauli, G., 2002. Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by realtime PCR. FEMS Microbiol. Lett. 214, 51–59. Heller, M.B., Bunning, M.L., France, M.E., Niemeyer, D.M., Peruski, L., Naimi, T., Talboy, P.M., Murray, P.H., Pietz, H.W., Kornblum, J., Oleszko, W., Beatrice, S.T., Joint Microbiological Rapid Response Team, New York City Anthrax Investigation Working Group, 2002. Laboratory response to anthrax bioterrorism, New York City, 2001. Emerg. Infect. Dis. 8, 1096–1102. Hoffmaster, A.R., Fitzgerald, C.C., Ribot, E., Mayer, L.W., Popovic, T., 2002a. Molecular subtyping of Bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, United States. Emerg. Infect. Dis. 8, 1111–1116. Hoffmaster, A.R., Meyer, R.F., Bowen, M.D., Marston, C.K., Weyant, R.S., Thurman, K., Messenger, S.L., Minor, E.E., Winchell, J.M., Rassmussen, M.V., Newton, B.R., Parker, J.T., Morrill, W.E., McKinney, N., Barnett, G.A., Sejvar, J.J., Jernigan, J.A., Perkins, B.A., Popovic, T., 2002b. Evaluation and validation of a real-time polymerase chain reaction assay for rapid identification of Bacillus anthracis. Emerg. Infect. Dis. 8, 1178–1182. Jernigan, J.A., Stephens, D.S., Ashford, D.A., Omenaca, C., Topiel, M.S., Galbraith, M., Tapper, M., Fisk, T.L., Zaki, S., Popovic, T., Meyer, R.F., Quinn, C.P., Harper, S.A., Fridkin, S.K., Sejvar, J.J., Shepard, C.W., McConnell, M., Guarner, J., Shieh, W.J., Malecki, J.M., Gerberding, J.L., Hughes, J.M., Perkins, B.A., Anthrax Bioterrorism Investigation Team, 2001. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg. Infect. Dis. 7, 933–944. Kiratisin, P., Fukuda, C.D., Wong, A., Stock, F., Preuss, J.C., Ediger, L., Brahmbhatt, T.N., Fischer, S.H., Fedorko, D.P., Witebsky, F.G., Gill, V.J., 2002. Large-scale screening of nasal swabs for Bacillus anthracis: descriptive summary and discussion of the National Institutes of Health's experience. J. Clin. Microbiol. 40, 3012–3016. Luna, V.A., King, D., Davis, C., Rycerz, T., Ewert, M., Cannons, A., Amuso, P., Cattani, J., 2003. Novel sample preparation method for safe and rapid detection of Bacillus anthracis spores in environmental powders and nasal swabs. J. Clin. Microbiol. 41, 1252–1255. Marston, C.K., Hoffmaster, A.R., Wilson, K.E., Bragg, S.L., Plikaytis, B., Brachman, P., Johnson, S., Kaufmann, A.F., Popovic, T., 2005. Effects of long-term storage on plasmid stability in Bacillus anthracis. Appl. Environ. Microbiol. 71, 7778–7780. Panning, M., Kramme, S., Petersen, N., Drosten, C., 2007. High throughput screening for spores and vegetative forms of pathogenic B. anthracis by an internally controlled real-time PCR assay with automated DNA preparation. Med. Microbiol. Immunol. (Berl.) 196, 41–50. Rotz, L.D., Khan, A.S., Lillibridge, S.R., Ostroff, S.M., Hughes, J.M., 2002. Public health assessment of potential biological terrorism agents. Emerg. Infect. Dis. 8, 225–230. Ryu, C., Lee, K., Yoo, C., Seong, W.K., Oh, H.B., 2003. Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR. Microbiol. Immunol. 47, 693–699. Sue, D., Marston, C.K., Hoffmaster, A.R., Wilkins, P.P., 2007. Genetic diversity in a Bacillus anthracis historical collection (1954 to 1988). J. Clin. Microbiol. 45, 1777–1782.
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Note
A simple method for the rapid removal of Bacillus anthracis spores from DNA preparations ☆ Leslie A. Dauphin ⁎, Michael D. Bowen Bioterrorism Rapid Response and Advanced Technology (BRRAT) Laboratory, Division of Bioterrorism Preparedness and Response (DBPR), National Center for Preparedness, Detection, and Control of Infectious Diseases (NCPDCID), Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA
a r t i c l e
i n f o
Article history: Received 8 October 2008 Accepted 9 October 2008 Available online 28 October 2008 Keywords: Bacillus anthracis Bacillus anthracis spores Anthrax Bioterrorism
a b s t r a c t This study establishes a filtration method for the safe removal of Bacillus anthracis spores which may contaminate DNA preparations. Centrifugal filtration with 0.1-μm filter units can be used following extraction of DNA from B. anthracis spores to render samples safe without compromising the sensitivity of diagnostic real-time PCR assays for B. anthracis. Published by Elsevier B.V.
Bacillus anthracis, the causative agent of anthrax, is regarded as one of the most effective biological weapons because of the potential for large-scale dissemination of spores (Rotz et al., 2002). In October 2001, the Ames strain of B. anthracis was used as a weapon of bioterrorism in the United States (Hoffmaster et al., 2002a; Jernigan et al., 2001). Since the 2001 anthrax attack, many nucleic acid amplification based assays for the detection of B. anthracis in clinical and environmental samples have been developed (Bell et al., 2002; Ellerbrok et al., 2002; Hoffmaster et al., 2002b; Ryu et al., 2003). The 2001 anthrax attack demonstrated that diagnostic laboratories are likely to receive large numbers of environmental specimens during a bioterrorism investigation (Heller et al., 2002; Kiratisin et al., 2002; Luna et al., 2003). Environmental specimens may contain spores, and it has been reported that most commercial extraction kits do not completely deplete B. anthracis spores from samples (Dauphin et al., 2008a; Panning et al., 2007). Studies have used centrifugal filter units during processing procedures for B. anthracis (Marston et al., 2005; Sue et al., 2007); however, none have established the optimal filter types and conditions for the elimination of spores from samples. The purpose of this study was to establish a rapid method for the complete removal of B. anthracis spores from DNA samples so that subsequent PCR testing can be carried out safely at lower levels of biocontainment. ☆ Disclaimer Statement: "This article reflects the findings and conclusions of the conducted research. Names of vendors or manufacturers are provided as examples of available product sources; inclusion does not imply endorsement of the vendors, manufacturers or products by the Centers for Disease Control and Prevention or the U.S. Department of Health and Human Services." ⁎ Corresponding author. BRRAT Laboratory, DBPR, NCPDCID, CDC, Mail Stop G-42, 1600 Clifton Road, Atlanta, GA 30333, USA. Tel.: +1 404 639 4991; fax: +1 404 639 4234. E-mail address: [email protected] (L.A. Dauphin). 0167-7012/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.mimet.2008.10.009
All procedures using virulent B. anthracis spores were performed in a biosafety level 3 laboratory. Eight strains of B. anthracis were used: K1340, K1694, K2478, K2484, K2762, K4596, K5135, and K9002. Spore suspensions were prepared using methods which have been described (Dauphin et al., 2008b). The Ultrafree-MC filter units in 1.5-ml tubes with microporous membranes and pore sizes of 0.1 μm, 0.22 μm, and 0.45 μm (Millipore Corporation, Billerica, MA) were evaluated for their ability to remove viable spores from samples as measured by performing colony counts of filtered sample extracts. A total of 100 filter units were tested for each pore size evaluated. A 100-μl aliquot of strain K1694 (Ames) spores at a concentration of 106 CFU/ml in phosphate-buffered saline (PBS) (0.01 M, pH 7.4) was loaded in each filter unit with great care so as to not puncture the filter membranes, and spore suspensions were centrifuged at 12,000 ×g for 2 min as recommended by the manufacturer. Immediately following filtration, the entire volume of each filtrate was spread onto Trypticase soy agar with 5% (vol/vol) sheep blood plates (TSAB) (BD Diagnostic Systems, Sparks, MD) and incubated for up to 48 h at 37 °C. Four commercial extraction kits were used: the ChargeSwitch gDNA Mini Bacteria Kit (Invitrogen, Carlsbad, CA), NucliSens Isolation Kit (BioMerieux, Inc., Durham, NC), Puregene Genomic DNA Purification Kit (Qiagen Inc., Valencia, CA), and QIAamp DNA Blood Mini Kit (Qiagen Inc., Valencia, CA). The kits were selected because it has been shown that sample extracts prepared from B. anthracis spores (≥105 CFU/ml) using these kits may be contaminated with viable spores (Dauphin et al., 2008a). A total of one hundred ninety-two samples were processed using the four kits, and the sample extracts were filtrated as described above. A 3-target (plasmid pXO1, plasmid pXO2, chromosome) real-time PCR assay (Hoffmaster et al., 2002b) was used to determine the effects
L.A. Dauphin, M.D. Bowen / Journal of Microbiological Methods 76 (2009) 212–214 Table 1 Comparison of centrifugal filter units for the removal of spores from B. anthracis spore suspensions Filter pore size (μm)a
Viable spores in filtrateb No. pos/no. tested
0.1 0.22 0.45
Confluent growth
b10 colonies
Efficiency (%)c
1/100 1/100 1/100
0/100 2/100 7/100
99 97 92
a Centrifugal filter units were evaluated using 100-μl aliquots of virulent B. anthracis Ames strain spores at a concentration of 106 CFU/ml, which were centrifuged at 12,000 ×g for 2 min. b The entire volume of the filtrates was plated onto Trypticase soy agar with 5% (vol/vol) sheep blood plates and viability was determined by direct observation of plates for colonies. Two different growth densities were observed on TSAB plates; confluency, and b 10 colonies. c The efficiency of the filter units was calculated by dividing the number of samples which contained no viable spores as measured by colony counts of filtered sample extracts, divided by the number of samples tested, multiplied by 100.
213
attributable to the centrifugation conditions. Since centrifugation at 6000 to 10,000 ×g for 2 min resulted in 100% removal of spores, these conditions were selected. To determine whether the filtration method affected the level of detection for B. anthracis DNA, real-time PCR results for filtered versus unfiltered DNA extracts were compared (Fig. 1). The filtration method did not significantly affect the detection levels for any of the three real-time PCR targets when compared by paired t test (two-tailed). The differences in mean CT values for the chromosomal target, 25.1 for filtered samples versus 24.7 for unfiltered samples, were not found to be significant (P = 0.53; n = 18, Fig. 1A), as were the differences in the mean CT values for the pXO1 target (24.1 and 23.6, respectively,
of the filtration method on the detection levels of B. anthracis DNA. Reactions were performed in 25-μl volumes including 5 μl of filtered or unfiltered sample extract using reagent concentrations, cycling conditions, and instrumentation as described previously (Dauphin et al., 2008b). Table 1 shows the results for viability testing of filtrates from B. anthracis Ames strain spores. Overall, filter units with a pore size of 0.1 μm were the most efficient at removing spores from samples. The efficiency of the 0.1-μm filter units was 99%, whereas the 0.22-μm and 0.45-μm filter units had efficiencies of 97% and 92%, respectively. Two different growth densities were observed on TSAB plates; confluency, and a few (b10) colonies. For each of the filter unit sizes, one sample filtrate out of 100 produced confluent growth on the TSAB plate suggesting that the centrifugation conditions resulted in a 1% failure rate for each pore size evaluated. In addition, two 0.22-μm filter unit filtrates, and seven 0.45-μm filter unit filtrates, produced growth of b10 colonies on TSAB plates indicating that for these samples, a few spores passed through the filter units in contrast to the confluent growth observed in cases of complete filter failure. To establish conditions which provide 100% removal of viable spores from samples, we further evaluated the 0.1-μm filter units. Table 2 shows the efficiency of 0.1-μm filter units under a range of centrifugation conditions. Centrifugation at 12,000 ×g for 1 to 2 min resulted in 99% efficiency, (1% failure rate characterized by confluent growth in culture) whereas centrifugation at 6000 to 10,000 ×g for 2 min consistently provided 100% removal of spores. These results suggest that the observed 1% failure rate for the 0.1-μm filter units was Table 2 Efficiency of 0.1-μm filter units for the removal of spores from B. anthracis spore suspensions under varying centrifugation conditions Conditionsa
Viable spores in filtrateb
Time (min)
Force (×g)
No. pos/no. tested
Efficiency (%)c
2.0 1.5 1.0 0.5 2.0 2.0 2.0
12,000 12,000 12,000 12,000 10,000 8000 6000
1/100 1/100 1/100 0/100 0/100 0/100 0/100
99 99 99 100 100 100 100
a
Centrifugation conditions were evaluated using 100-μl aliquots of live B. anthracis Ames strain spores at a concentration of 106 CFU/ml. b The entire volume of the filtrates was plated onto Trypticase soy agar with 5% (vol/vol) sheep blood plates and viability was determined by direct observation of plates for colonies. All filtrates which were positive for viable spores resulted in confluent growth in culture. c The efficiency for the removal of viable spores was calculated by dividing the number of samples which contained no viable spores as measured by colony counts of filtered sample extracts, divided by the number of samples tested, multiplied by 100.
Fig. 1. Results of TaqMan real-time PCR analysis of filtered versus unfiltered sample extracts. DNA was prepared from virulent B. anthracis Ames strain spores at a concentration of 106 CFU/ml using the QIAamp kit and assayed using the (A), BA3 chromosome-specific primer/probe set; (B), BA2 pXO1 plasmid-specific primer/probe set; and (C), BA1 pXO2 plasmid-specific primer/probe set. The results were plotted as the mean relative fluorescence for filtered and unfiltered sample extracts, and no template controls (NTC) versus the cycle numbers. The differences in CT values between filtered versus unfiltered samples were not found to be significant using a paired t test (two tailed) for the chromosome (P = 0.53; n = 18), pXO1 plasmid (P = 0.52; n = 18) and pXO2 plasmid (P = 0.46; n = 18) targets.
214
L.A. Dauphin, M.D. Bowen / Journal of Microbiological Methods 76 (2009) 212–214
P = 0.52; n = 18, Fig. 1B) and the pXO2 target (24.7 and 23.8, respectively, P = 0.46; n = 18, Fig. 1C). Further, the method was tested on triplicate DNA samples prepared from the spores of eight strains of B. anthracis using four commercial extraction kits and the results indicated 100% removal of viable spores in 0.1 volume test aliquots. This study established a filtration method for the rapid removal of B. anthracis spores from DNA preparations without significantly affecting the detection of B. anthracis DNA in real-time PCR. Centrifugal filtration with 0.1-μm filter units at 6000 to 10,000 ×g for 2 min can be used by diagnostic laboratories involved in testing of environmental samples during investigations of suspected events of anthrax bioterrorism to rapidly remove spores from DNA samples prior to testing. Safety testing by culture is the only way to guarantee that DNA extracts are free of infectious spores but in situations when time-to-results is critical and the testing laboratory cannot wait 24 to 48 h for safety testing results, the method developed in this study will significantly reduce the frequency of spore contamination in DNA extracts. We thank John Ridderhof, Pamela Diaz, Harvey Holmes, Patricia Fields, Bruce Newton, Heather Stang, Justin Schrager, Rebecca Hutchins, and Kenyatta Stephens for their critical review of this manuscript. B. anthracis is a select agent and its possession, use, and transfer is regulated by the U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, and the U.S. Department of Agriculture, Animal and Plant Health Inspection Service. The select agent regulations have mandatory reporting requirements for identification of select agents in diagnostic specimens. References Bell, C.A., Uhl, J.R., Hadfield, T.L., David, J.C., Meyer, R.F., Smith, T.F., Cockerill III, F.R., 2002. Detection of Bacillus anthracis DNA by LightCycler PCR. J. Clin. Microbiol. 40, 2897–2902. Dauphin, L.A., Moser, B.D., Bowen, M.D., 2008a. Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus anthracis spores and comparison of DNA yields from spores and spiked environmental samples. J. Microbiol. Methods. doi:10.1016/j.mimet.2008.09.004. Dauphin, L.A., Newton, B.R., Rasmussen, M.V., Meyer, R.F., Bowen, M.D., 2008b. Gamma irradiation can be used to inactivate Bacillus anthracis spores without compromising the sensitivity of diagnostic assays. Appl. Environ. Microbiol. 74, 4427–4433.
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