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Evaluation of DNA extraction methods for Bacillus anthracis spores spiked to food and feed matrices at biosafety level 3 conditions
International Journal of Food Microbiology 150 (2011) 122–127
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International Journal of Food Microbiology 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 / i j f o o d m i c r o
Evaluation of DNA extraction methods for Bacillus anthracis spores spiked to food and feed matrices at biosafety level 3 conditions Peter R. Wielinga a,⁎, Lianne de Heer a, Astrid de Groot a, Raditijo A. Hamidjaja a, Geert Bruggeman b, Kieran Jordan c, Bart J. van Rotterdam a a National Institute for Public Health and the Environment (RIVM), Centre for Infectious Disease Control (CIb), Laboratory for Zoonoses and Environmental Microbiology (LZO), Antonie van Leeuwenhoeklaan 9, P.O. Box 1, Bilthoven, The Netherlands b Nutrition Sciences N.V., Vitamex Group, Belgium c Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
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Article history: Received 27 September 2010 Received in revised form 25 May 2011 Accepted 21 July 2011 Available online 29 July 2011 Keywords: Quantitative multiplex real-time PCR Bacillus anthracis Bacillus thuringiensis Anthrax DNA extraction efficiency PCR inhibitors
a b s t r a c t The DNA extraction efficiency from milk, whey, soy, corn gluten meal, wheat powders and heat-treated corn grain that were spiked with Bacillus anthracis and Bacillus thuringiensis spores was determined. Two steps were critical: lysis of the spores and binding of the free DNA to the DNA binding magnetic beads in the presence of the interfering powders. For the guanidine-thiocyanate based Nuclisens lysis buffer from Biomerieux we found that between 15 and 30% of the spores survived the lysis step. As most lysis buffers in DNA/RNA extraction kits are guanidine based it is likely that other lysis buffers will show a similar partial lysis of the Bacillus spores. Our results show that soybean flour and wheat flour inhibited the DNA extraction process strongest, leading to unreliable DNA extractions when using too much of the matrix. For corn gluten meal, heat-treated corn grain and milk powders, DNA extraction efficiencies in the presence of 100 mg and 10 mg of powder resulted in 70%–95% reduced DNA recoveries. The inhibition was, however, reliable and intermediate compared to the inhibition by soy and wheat. Whey powder had the lowest inhibitory effect on DNA-extraction efficiency and recoveries of 70–100% could be reached when using 10 mg of powder. The results show that reducing the amount of matrix leads to better DNA-extraction efficiencies, particularly for strongly inhibiting powders such as soy and wheat. Based on these results, a standard protocol to directly isolate DNA from micro-organisms present in complex matrixes such as food and feed powders was designed. © 2011 Elsevier B.V. All rights reserved.
1. Introduction For detection of contaminating micro-organisms in food and feed, PCR is becoming the method of choice (Malorny et al., 2003; Settanni and Corsetti, 2007). For food and feed samples, adding a (diluted) sample directly to a PCR reaction is for most cases not possible due to the presence of PCR inhibitors in the sample. For PCR based detection of such samples, one first needs to either enrich the culture prior to analysis or to extract the total DNA directly from the sample. Both methods have advantages and disadvantages. Culturing may give pure cultures which makes detection easy and makes DNA-extraction unnecessary. It has the disadvantage though of being time consuming, it is difficult to show a specific contamination when different types of micro-organisms are present, it is not generic in the sense that specific micro-organisms require specific growth conditions and quantification is difficult after enrichment. Direct extraction of total DNA has the advantage of being fast, generic and unbiased towards the DNA being
⁎ Corresponding author. Tel.: + 31 30 2747034; fax: + 31 30 2744434. E-mail address: [email protected] (P.R. Wielinga). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.07.023
extracted. The disadvantages include lack of sensitivity, the extraction of DNA from dead cells and inhibiting effects of the matrix from which the microbial DNA has to be extracted. Bacillus anthracis is part of the B. cereus family, consisting of the genetically closely related members: B. cereus, B. thuringiensis and B. anthracis (Bavykin et al., 2004). All three members form spores and are found world-wide. Their spores are highly resistant to different kinds of stress, enabling them to survive harsh environmental conditions (Driks, 2003), including some food processing procedures (Khan et al., 2009). B. anthracis is a zoonotic pathogen that may cause life threatening diseases in both animals and humans (Dixon et al., 1999; Mock and Fouet, 2001). After the anthrax letter attack in the US in 2001 (Jernigan et al., 2001), governments, the scientific community and the food and feed industries realised that in order to prevent a recurrence, development of simple and fast protocols for detecting highly dangerous pathogens (e.g. B. anthracis) is essential for an adequate and timely response to acts of bioterrorism. Since 2001, many different rapid methods have been developed and published for detection of DNA from biothreat agents such as B. anthracis, Francisella tularensis, Clostridium botulinum and Yersinia pestis (Antwerpen et al., 2008; Easterday et al., 2005; Fach
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et al., 2010; Hadjinicolaou et al., 2009; Skottman et al., 2007; Van Ert et al., 2007; Wielinga et al., 2010; Wilson et al., 2005). The essential step prior to this, DNA-extraction, however, has received less attention. There have been studies comparing different DNA extraction kits which focussed on using pure cultures or DNA. However, when extracting DNA directly from various matrices, the matrix can influence the efficiency of DNA extraction as well. The role of food and feed in a possible act of bioterrorism is a distinct possibility as shown by the use of infected food products in the past as a vector of intentional contamination (Anderson et al., 1996; Byrne et al., 2006; Hanson et al., 2005; Jernigan et al., 2001; Wein and Liu, 2005; see also the review by Keim and Wagner, 2009). Thus, contaminated products may need to be investigated. During such a bioterroristic event, PCR (involving extraction and detection) may be used to screen samples rapidly. Developing methodologies in preparation for such an event is important. In order to achieve this in a reliable manner, it is essential to understand the negative effects that different matrices may have on DNA-extraction, and take these into account when designing a suitable PCR detection protocol for these matrices. For the detection of a wide variety of so-called select agents these protocols need to be validated and applied under biosafety level 3 (BSL-3) or even higher laboratory safety conditions (Richmond and Nesby-O'Dell, 2002). This involves working under strict and controlled conditions, such as in an air tight pressure controlled glove box in a pressure controlled laboratory. This complicates the working procedures to a great extent and demands skilled and trained laboratory workers. It also demands well documented and validated working procedures which have been validated and are performed under the guidance of a biosafety officer. In previous studies, several different DNA-extraction kits were tested for DNA-extraction from different micro-organisms including Y. pestis, F. tularensis and B. anthracis (Coyne et al., 2004; Dauphin et al., 2009a,b; Miller et al., 1999; Ramisse et al., 1999; Sagova-Mareckova et al., 2008; Whitehouse and Hottel, 2007). Comparing six different DNA-extraction kits, the NucliSens kit was shown to have the most favourable characteristics (Dauphin et al., 2009a). We therefore chose to study this DNA-extraction kit further in our experiments. This kit uses a guanidine-thiocyanate solution which is a highly chaotropic chemical solution, and it is assumed that this type of medium results in complete lysis of all cell types, including spores. However, it has been shown that a certain fraction of spores can survive this harsh treatment (Miller et al., 1999; Dauphin and Bowen, 2009). This increases the challenge of extracting DNA from spores prior to PCR-detection. In this study, B. thuringiensis spores were initially used as a model organism for B. anthracis. Different food and feed powders were intentionally contaminated and the DNA-extraction efficiency of the NucliSens kit was studied under normal laboratory conditions. Furthermore, we determined to what extent PCR inhibitors were coextracted. Using the results from these experiments, we formulated an optimal protocol and tested this under BSL-3 conditions using 10 different food and feed powders spiked with B. anthracis spores. 2. Materials and methods 2.1. Food and feed samples Corn gluten meal, whey powder, wheat flour, soybean flour and heat-treated corn grain were supplied by Vitamex N.V. (Belgium). All these raw materials are used as major basic compounds for animal feed. The Irish milk and whey powders were from Dairygold Cooperative Ltd, Mitchelstown, Cork, Ireland. Low, medium and high heat skim milk powders (SMP) were manufactured by preheating milk to 72 °C × 12 s, 97 °C × 2 min and 120 °C × 2 min, respectively, prior to evaporation and drying. Typically, the powders contain 33% protein and 55% lactose. Whey powder was manufactured by evaporation and drying of cheese-whey. Typically, whey powder contains 12% protein
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and 75% lactose. Whey protein concentrate (WPC) was manufactured by ultrafiltration of whey to 35% protein prior to evaporation and drying of the retentate. Typically, WPC35 contains 35% protein and 55% lactose. 2.2. Polymerase chain reaction (PCR) Quantitative real time PCR (qPCR) reactions for B. anthracis and B. thuringiensis were conducted in multiplex format in 20 μl reaction mixtures using iQ Multiplex Powermix (Biorad) and primers and probes as described previously (Wielinga et al., 2010). The PCR was carried out under the following conditions: 5 min of denaturation at 95 °C followed by 50 cycles of 5 s 95 °C and 35 s 60 °C (amplification and detection) on an LC480 (Roche). The threshold cycle value (Ctvalue) at which a statistically significant increase of the fluorescence signal is first detected was determined by the second derivative method of the LightCycler 480 Software release 1.5.0. SP3 (Roche). 2.3. Strains and cultures The B. anthracis and B. cereus strains have been described previously (Carlin et al., 2006; Wahab et al., 2005). B. thuringiensis ATCC29730 spores were purchased from Raven Biological Laboratories (Omaha, Nebraska, USA). Vegetative cells were grown under aerobic conditions in brain hearth infusion (BHI) medium (Gibco) at 37 °C. Spores were generated by growing in sporulation medium. Sporulation was determined by comparing growth of untreated and heat treated (15 min 65 °C) cells on BHI agar plates and growing overnight at 30 °C. For B. cereus and B. thuringiensis, microscopic inspection confirmed that of the suspensions used more than 99% of the cells had sporulated. All handling of vegetative B. anthracis cells and spores was performed under BSL-3 conditions. 2.4. DNA extraction Cell lysis and DNA extractions were performed using NucliSens lysis buffer and NucliSens DNA Magnetic Extraction Reagents (Biomerieux, Boxtel, The Netherlands). To determine the DNA-extraction efficiency from spores in the presence of different powders under normal laboratory conditions, 100 mg powder was directly added to 10 mL of lysis buffer and spiked with 3 × 105 B. thuringiensis spores. After incubating and mixing (manually) for 10 min with magnetic silica particles the bead-bound DNA was recovered according to the manufacturer's instructions and the B. thuringiensis DNA-concentration was determined using a multiplex qPCR which detects both B. thuringiensis and B. anthracis DNA (Wielinga et al., 2010). To determine the extent of possible co-extracted PCR-inhibitors, the DNA extraction samples were spiked with B. anthracis DNA and compared to the measured Ct-values of non-powder extraction controls. Since diluting out possible inhibitors sometimes leads to a better PCRdetection we determined the Ct-values for both the undiluted and 10times diluted extracted DNA samples. For each sample the Ct-values were determined in duplicate. The percentage of DNA recovery was calculated relative to the control (set at 100%) using the comparative Ctmethod (Pfaffl, 2001; Livak, 2001) and a PCR efficiency of 2, using: Recovery (%) = 100%·[2 (Ct, sample − Ct, control)] −1. For each of the individual experiments the recovery was calculated, after which the average recovery was calculated by taking the mean of these values. Statistical significance was calculated using a Student's t-test; a value of p b 0.05 was considered significantly different from the control extraction. For B.anthracis, extractions were performed under BSL-3 conditions, adding 10 mg of powder to 10 mL of lysis buffer, spiking with 3 × 105 B. anthracis spores and extracting the DNA. Before removal from the isolator, all the DNA samples were filtered through a 0.22 μm Ultrafree Centrifugal Filter Unit (Millipore) to remove surviving spores.
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2.5. Survival of cells and spores in lysis buffer Spores and vegetative cells of either B. thuringiensis or B. cereus spores (10 6/mL) were incubated for 10 min in medium containing a decreasing percentage of lysis buffer. After incubation, 10-fold dilution series were made in BHI-medium supplemented with 0.05% TritonX100, the dilutions plated on BHI agar plates and grown overnight at 30 °C to determine the number of colony forming units (CFUs). 3. Results and discussion 3.1. Spore viability Spore viability was tested by determining the number of CFUs before and after incubation for 15 min at 65 °C. For both the B. cereus and B. thuringiensis spores, similar counts were obtained before and after 15 min heat treatment at 65 °C, showing a sporulation efficiency approaching 100%. Microscopic examination confirmed the sporulation efficiency of 99%–100%. Similar heat treatments of vegetative cells reduced the CFU count to zero. The efficiency of spore lysis by the NucliSens lysis buffer was assessed to determine if some spores survived this treatment. Fig. 1 shows the percentage of surviving B. cereus and B. thuringiensis spores and vegetative cells at decreasing lysis buffer concentrations. Treatment of vegetative cells with 100% lysis buffer led to a 100% reduction in CFUs. Treatment of B. cereus and B. thuringiensis spores with 100% lysis buffer led to 85% and 70% reduction in CFUs, respectively. This indicated that between 15% and 30% of the spores survived the lysis treatment and that their DNA was unavailable for extraction, which is important informa-
tion when PCR is being considered for screening purposes. In addition, this showed that although the sporulation level approached 100% and all spores appeared microscopically identical, there was a fraction of the spores withstanding the harsh treatment of the lysis buffer. 3.2. Direct DNA extraction from spores in the presence of 100 mg food and feed powders To determine the DNA extraction efficiency from spores in the presence of 10 different powders that are used in the food and feedindustry, 100 mg powder was spiked with 3 × 10 5 B. thuringiensis spores and total DNA was extracted. Table 1 shows the recovery (in %) of B. thuringiensis DNA and the PCR efficiency of the internal PCR control for ten different milk, whey, soy, wheat, corn and wheat powders, for both the undiluted and a 10-times diluted DNA extraction sample. Soybean flour and wheat flour inhibit the DNA-extraction process the most, followed by corn gluten meal, heat-treated corn grain and milk powders giving a relative intermediate inhibition. Whey powder was only mildly inhibitory. Comparing the undiluted and 10-times diluted samples showed that diluting the samples improved the sensitivity of B. thuringiensis DNA detection for several powders. For the undiluted samples only the whey powders showed a relatively good recovery (22% and 44%). Wheat flour and in particular the soybean flour showed very low recoveries. At a 10-fold dilution the recovery increased strongly for heat-treated corn grain, wheat, soy and corn gluten meal (see Table 1). For soy and corn gluten meal, this increase was also due to diluting out co-extracted PCR inhibitors, as the internal control detection efficiency went from 55% and 42% for the undiluted samples to about 100% for the 10-fold diluted sample (Table 1, compare fourth and fifth column). Further comparison of the internal control detection efficiencies showed that for the other the powders the PCR was not greatly affected by possible co-extracted substances. 3.3. Optimization of the direct DNA extraction protocol
Fig. 1. Percentage lysis of cells and spores in lysis buffer. After incubating vegetative cells (squares) and spores (diamonds) of B. thuringiensis (A) or B. cereus (B) for 10 min in the indicated percentage of lysis buffer, the colony forming units (CFU) were determined.
For both soybean flour and wheat flour, representing the two most interfering matrices, we optimised the extraction and determined the relationship between DNA recovery and several physical parameters. The relationship between DNA recovery and the amount of soybean flour and wheat flour added to 10 mL lysis buffer is shown in Fig. 2A. This shows that at concentrations above 10 mg powder per 10 mL lysis buffer, the DNA recovery was strongly inhibited and approaches 0%. At lower powder concentrations, the recovery increased exponentially and at 10 mg powder per 10 mL lysis buffer, the DNA could be recovered reproducibly from both wheat and soybean flour. Fig. 2B shows the relationship between DNA recovery and volume of lysis buffer used, while keeping the amount of soybean flour and incubation time constant. This shows that increasing the volume up to 50 mL led to a substantial decrease (almost 60%) in DNA recovery for the control sample (without powder added), which was due to dilution of the DNA. In contrast, and in spite of this decrease, recovery in the presence of 20 mg soybean flour was increased from almost 0% to 1.5%. This shows that decreasing the inhibition by lowering the powder concentration over-compensates the decreasing DNA concentration. Together, this counter intuitively shows that by lowering the amount of a contaminated powder in a DNA extraction one may increase ones chance of detecting the contamination, in particular when handling strongly inhibiting powders. Fig. 3 shows the relationship between DNA recovery and the incubation time in lysis buffer. For the no-powder control, maximum DNA binding occurred between 5 and 15 min. In the presence of wheat flour, recovery was maximal at 5 min and decreased over time. This indicates that, next to the DNA binding process other, as yet unknown, processes take place during lysis that make it disadvantageous to incubate for longer periods. For soybean flour, DNA recovery increased
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Table 1 The DNA recovery and the internal control detection efficiency for ten different powders (100 mg powder/10 mL lysis buffer) spiked with B. thuringiensis (3 × 105) spores. Sample
No addition control Corn gluten meal (Belgium) Whey powder (Belgium) Wheat flour (Belgium) Soybean flour (Belgium) Heat-treated corn grain (Belgium) SMP high heatc (Ireland) SMP medium heat (Ireland) SMP low heat (Ireland) WPC 35 UFd (Ireland) Whey powder (Ireland)
DNA recovery (%)
a
Internal control detection efficiency (%)
b
Undiluted
1 to 10
Undiluted
1 to 10
100% 6.3% ± 5.4% (29.2) 22% ± 14% (27.3) 0.7% ± 0.4% (37.5) 0.01% (37.0)e 3.7% ± 3.3% (30.4) 3.7% ± 0.2% (30.4) 4.2% ± 2.1% (30.1) 3.6% ± 2.9% (30.5) 5.1% ± 0.3% (29.6) 44% ± 15% (26.7)
100% 43% ± 72% (24.3) 60% ± 62% (24.1) 29% ± 45% (24.7) 2.1% ± 1.7% (29.6) 22% ± 27% (24.9) 4.0% ± 1.5% (27.8) 2.8% ± 0.4% (28.8) 2.6% ± 1.2% (29.0) 3.8% ± 1.7% (27.9) 38% ± 11% (24.4)
100% 90% ± 13% (29.1) 111% ± 16% (28.9) 121% ± 13% (28.9) 55% ± 50% (29.4) 42% ± 47% (29.5) 91% ± 7.0% (29.1) 117% ± 27% (28.9) 108% ± 19% (29.0) 149% ± 59% (28.8) 116% ± 29% (28.9)
100% 107% ± 14% (26.5) 112% ± 13% (26.4) 116% ± 25% (26.4) 103% ± 12% (26.5) 115% ± 11% (26.4) 105% ± 11% (26.5) 118% ± 8.0% (26.4) 119% ± 5.9% (26.4) 112% ± 24% (26.4) 107% ± 11% (26.5)
a For each extraction the Ct-value was determined in duplicate for the undiluted as well as a 10-times diluted sample. The DNA recovery (in %) was calculated relative to the control (set at 100%) using the comparative Ct-method (Pfaffl, 2001; Livak, 2001) using a PCR efficiency of 2: Recovery (%) = 100%·[2(Ct, sample − Ct, control)]−1. Data are given as the mean of n = 3 ± SD, except for the Irish powders which are given as the mean of n = 2 ± the average difference. Between brackets the average Ct-value is given for reference. The underlined values were not significantly different from the no powder extraction control calculated using a Student's t-test (p N 0.05). b As internal control, the DNA-extraction samples were spiked with B. anthracis DNA and detection efficiency calculated relative to the control (B. anthracis DNA alone). c Three different fractions of skim milk powders (SMP) were tested. d WPC — Whey Protein Concentrate 35% obtained by ultrafiltration. e For soybean flour two experiments resulted in a complete loss of PCR signal, while for the third experiment a weak curve with a Ct-value of 37 was observed.
over time and after 60 min the maximum was not reached, suggesting that longer incubation may increase the recovery even more. 3.4. Performance of DNA extraction method for B. anthracis spores under BSL-3 conditions From the data shown in Table 1 and Figs. 1–3 an optimised DNA extraction protocol that balanced the amount of powder, incubation time and volume of lysis buffer used, was formulated. The optimised protocol used 10 mL lysis buffer, 10 mg powder and 10 min lysis and DNA binding to the DNA-binding magnetic beads. This protocol was tested for the extraction of DNA from B. anthracis spores under BSL-3 conditions. Table 2 shows the recovery of B. anthracis from the powders tested (spiked with 3 × 10 5 B. anthracis spores). These data show that using this protocol, B. anthracis was reliably detected in all powders. Comparing the extraction efficiencies of the undiluted samples (Tables 1 and 2) showed that the 10-fold reduction of matrix increased the recovery for all powders tested, except for heat-treated corn grain. For soybean flour and wheat flour this gain was most evident. For these powders, the recovery went from 0.01% and 0.7% at 100 mg to 2% and 10% at 10 mg, respectively. For heat-treated corn grain, the recovery decreased from 3.7% to 0.5%, in contrast to what was expected. The reason for this is not clear and may be due to co-
Fig. 2. Effect of the amount of powder and the amount of lysis buffer on the DNA extraction efficiency. A) B. thuringiensis spores were incubated for 10 min at room temperature in 10 mL lysis buffer in the presence of the indicated amounts of soybean flour (diamonds) and wheat flour (squares) powder. Thereafter, the DNA was extracted and the amount determined by qPCR. The extraction efficiency was calculated relative to the control (no powder added) and is given as the average of n = 2 determinations (± the average difference). B) B. thuringiensis spores were incubated for 10 min with the indicated amount of lysis buffer in the presence (diamonds) and absence (squares) of 20 mg soybean flour. The extraction efficiency was calculated relative to the control (no powder added) determent with 10 mL lysis buffer.
Fig. 3. Effect of the incubation time on the DNA extraction efficiency. B. thuringiensis spores were incubated with soybean flour (squares) and wheat flour (triangles) for the indicated periods of time after which the DNA was extracted. The extraction efficiency was calculated and plotted relative to the amount of B. thuringiensis DNA extracted after 60 min for the no powder control (circles).
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Table 2 DNA extraction efficiencies under BSL-3 conditions. Powders (10 mg powder/10 mL lysis buffer) were spiked with 3 × 105 B. anthracis and incubated of 10 min in the presence of DNA binding beads after which DNA was isolated. Sample
No addition control Corn gluten meal (Belgium) Whey powder (Belgium) Wheat flour (Belgium) Soybean flour (Belgium) Heat-treated corn grain (Belgium) SMPb high heat (Ireland) SMP medium heat (Ireland) SMP low heat (Ireland) WPC 35 UFc (Ireland) Whey powder (Ireland)
B. anthracis spore DNA recovery (%)a Undiluted
1 to 10
100% 18% ± 12% (32.4) 70% ± 32% (31.2) 10% ± 7.7% (33.2) 2.0% ± 3.1% (37.0) 0.5% ± 1.1% (44.7) 34% ± 54% (31.7) 48% ± 80% (31.4) 17% ± 24% (32.5) 64% ± 52% (31.3) 100% ± 30% (31.0)
100% 25% ± 24% (34.7) 70% ± 43% (33.9) 13% ± 7.9% (35.5) 11% ± 11% (35.8) 16% ± 23% (35.2) 47% ± 77% (34.2) 58% ± 108% (34.0) 21% ± 37% (34.9) 67% ± 71% (33.9) 99% ± 64% (33.7)
a For each extraction the Ct-value was determined in duplicate for the undiluted as well as 10-times diluted sample. The extraction efficiencies were calculated as described in Table 1. Data are given as the mean of n = 3 ± SD. Between brackets the average Ct-value is given for reference. The underlined values were not significantly different from the no powder extraction control calculated using a Student's t-test (p N 0.05). b Three different fractions of skim milk powders (SMP) were tested. c WPC — Whey Protein Concentrate 35% obtained by ultrafiltration.
extracted B. anthracis PCR specific inhibitor. This is, however, not very likely as the two different organisms, B. thuringiensis and B. anthracis, for which the spores may react differently we assume it unlikely that their DNA reacts differently in these extractions. More relevant was the observation that in the lysis buffer suspension this powder was difficult to separate from the magnetic silica particles, which is particularly challenging under BSL-3 conditions. Diluting the extracted DNA before PCR detection to reduce the effects of co-extracted PCR inhibitors led to a strong gain in DNA recovery for heat-treated corn grain as well as for soybean flour. This indicated that a substantial amount of PCR inhibiting substance was still co-extracted from the 10 mg of these powders. For the other powders, diluting the DNA extract led to a small gain in the recovery, which indicated that at 10 mg no substantial amount of inhibiting substances was extracted from these powders. Our work shows that the detection method we present here can be satisfactorily applied in a BSL-3 laboratory for examination of food and feed powders potentially contaminated with B. anthracis. When using matrices other than the ones used here, it is advisable to test what effect these matrices have on DNA extraction before starting large scale laboratory work at BSL-3 or BSL-4 level. Testing a range between 100 and 5 mg may be good starting point for this; weighing less than 5 mg of material is complicated and may become unreliable under BSL-3 conditions. In the case of an emergency, for instance, when one assumes a bioterrorist attack, one needs to move swiftly. For such cases or when one is limited by the amount or knowledge of the sample material our optimised protocol may be used in a relatively reliable manner: use maximally 10 mg powder per 10 mL lysis buffer and determine the DNA concentration for both an undiluted and a 10-fold diluted extraction sample, preferentially using an internal extraction control. In the case of bulk volumes that need to be tested, one might scale up this amount while keeping the concentration of powder in the lysis buffer the same, or more practically when working under BSL-3 conditions, one may increase the sample size. 4. Concluding remarks We determined the spore DNA-extraction efficiency from ten different milk, whey, soy, corn and wheat powders spiked with B. anthracis and B. thuringiensis spores. We found that the critical steps in DNA-extractions are both lysis of the spores and binding of the DNA to the beads in the presence of the interfering powders. We showed
that between 15 and 30% of the spores survived the lysis step and thus their DNA was lost for detection. For our experiments, we used NucliSens lysis buffer, which is a guanidine thiocyanate based lysis buffer. Guanidine thiocyanate is a chemical which is generally used in many different lysis buffer formulations. Therefore, we think it is likely that for similar lysis buffers, in other DNA isolation kits, a similar partial lysis of the Bacillus spores may occur. We showed that (when using too much of the matrix) soybean flour and wheat flour inhibited the DNA-extraction process strongly, leading to unreliable DNA-extractions. For corn gluten meal, heattreated corn grain and milk powder the extractions at 100 mg and 10 mg powder resulted in an approximately 70%–95% reduced DNA recovery. Their inhibition was, however, reliable and intermediate when compared to the other powders tested. For whey powder, the DNA extractions were most reliable with DNA extraction efficiencies between 70 and 100% when using 10 mg powder and 20–40% when using 100 mg powder, respectively. The goal of our study was to report an optimised strategy for a general direct DNA extraction procedure for Bacillus spores in food and feed powders. Our experiments showed that for some strongly interfering powders, apparently logical approaches did not give the expected improved response. For such powders, adding more powder with a small trace of infectious agent did not lead to better, but to worse recoveries. From this, we conclude that when one is unfamiliar with the properties of a powder, it may be better to use less powder to be sure of extracting some DNA. In addition, our experiments showed that the incubation time of the powder in lysis buffer with the DNA-binding beads differs per powder and needs to be optimised. However, when one is unfamiliar with the exact matrix/powder, an incubation time of about 10–15 min seems best. Acknowledgement This work was supported by the European Union-funded Integrated Project BIOTRACER (contract #036272) under the 6th RTD Framework Programme of the European Union. References Anderson, R.M., Donnelly, C.A., Ferguson, N.M., Woolhouse, M.E., Watt, C.J., Udy, H.J., MaWhinney, S., Dunstan, S.P., Southwood, T.R., Wilesmith, J.W., Ryan, J.B., Hoinville, L.J., Hillerton, J.E., Austin, A.R., Wells, G.A., 1996. Transmission dynamics and epidemiology of BSE in British cattle. Nature 382, 779–788. Antwerpen, M.H., Zimmermann, P., Bewley, K., Frangoulidis, D., Meyer, H., 2008. Realtime PCR system targeting a chromosomal marker specific for Bacillus anthracis. Molecular and Cellular Probes 22, 313–315. Bavykin, S.G., Lysov, Y.P., Zakhariev, V., Kelly, J.J., Jackman, J., Stahl, D.A., Cherni, A., 2004. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms. Journal of Clinical Microbiology 42, 3711–3730. Byrne, B., Dunne, G., Bolton, D.J., 2006. Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll. Food Microbiology 23, 803–808. Carlin, F., Fricker, M., Pielaat, A., Heisterkamp, S., Shaheen, R., Salonen, M.S., Svensson, B., Nguyen-the, C., Ehling-Schulz, M., 2006. Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group. International Journal of Food Microbiology 109, 132–138. Coyne, S.R., Craw, P.D., Norwood, D.A., Ulrich, M.P., 2004. Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit. Journal of Clinical Microbiology 42, 4859–4862. Dauphin, L.A., Bowen, M.D., 2009. A simple method for the rapid removal of Bacillus anthracis spores from DNA preparations. Journal of Microbiological Methods 76, 212–214. Dauphin, L.A., Moser, B.D., Bowen, M.D., 2009a. 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. Journal of Microbiological Methods 76, 30–37. Dauphin, L.A., Stephens, K.W., Eufinger, S.C., Bowen, M.D., 2009b. Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples. Journal of Applied Microbiology 108, 163–172. Dixon, T.C., Meselson, M., Guillemin, J., Hanna, P.C., 1999. Anthrax. The New England Journal of Medicine 341, 815–826.
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Ramisse, V., Patra, G., Vaissaire, J., Mock, M., 1999. The Ba813 chromosomal DNA sequence effectively traces the whole Bacillus anthracis community. Journal of Applied Microbiology 87, 224–228. Richmond, J.Y., Nesby-O'Dell, S.L., 2002. Laboratory security and emergency response guidance for laboratories working with select agents. Centers for Disease Control and Prevention. Morbidity and Mortality Weekly Report (MMWR) Recommendations and Reports 51, 1–6. Sagova-Mareckova, M., Cermak, L., Novotna, J., Plhackova, K., Forstova, J., Kopecky, J., 2008. Innovative methods for soil DNA purification tested in soils with widely differing characteristics. Applied and Environmental Microbiology 74, 2902–2907. Settanni, L., Corsetti, A., 2007. The use of multiplex PCR to detect and differentiate foodand beverage-associated microorganisms: a review. Journal of Microbiological Methods 69, 1–22. Skottman, T., Piiparinen, H., Hyytiainen, H., Myllys, V., Skurnik, M., Nikkari, S., 2007. Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis. European Journal of Clinical Microbiology and Infectious Diseases 26, 207–211. Van Ert, M.N., Easterday, W.R., Huynh, L.Y., Okinaka, R.T., Hugh-Jones, M.E., Ravel, J., Zanecki, S.R., Pearson, T., Simonson, T.S., U'Ren, J.M., Kachur, S.M., LeademDougherty, R.R., Rhoton, S.D., Zinser, G., Farlow, J., Coker, P.R., Smith, K.L., Wang, B., Kenefic, L.J., Fraser-Liggett, C.M., Wagner, D.M., Keim, P., 2007. Global genetic population structure of Bacillus anthracis. PloS One 2, e461. doi:10.1371/journal. pone.0000461. Wahab, T., Hjalmarsson, S., Wollin, R., Engstrand, L., 2005. Pyrosequencing Bacillus anthracis. Emerging Infectious Diseases 11, 1527–1531. Wein, L.M., Liu, Y., 2005. Analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk. Proceedings of the National Academy of Sciences of the United States of America 102, 9984–9989. Whitehouse, C.A., Hottel, H.E., 2007. Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples. Molecular and Cellular Probes 21, 92–96. Wielinga, P.R., Hamidjaja, R.A., Agren, J., Knutsson, R., Segerman, B., Fricker, M., EhlingSchulz, M., de Groot, A., Burton, J., Brooks, T., Janse, I., van Rotterdam, B., 2010. A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types. International Journal of Food Microbiology (Supplement 1), S137–S144. Wilson, W.J., Erler, A.M., Nasarabadi, S.L., Skowronski, E.W., Imbro, P.M., 2005. A multiplexed PCR-coupled liquid bead array for the simultaneous detection of four biothreat agents. Molecular and Cellular Probes 19, 137–144.
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International Journal of Food Microbiology 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 / i j f o o d m i c r o
Evaluation of DNA extraction methods for Bacillus anthracis spores spiked to food and feed matrices at biosafety level 3 conditions Peter R. Wielinga a,⁎, Lianne de Heer a, Astrid de Groot a, Raditijo A. Hamidjaja a, Geert Bruggeman b, Kieran Jordan c, Bart J. van Rotterdam a a National Institute for Public Health and the Environment (RIVM), Centre for Infectious Disease Control (CIb), Laboratory for Zoonoses and Environmental Microbiology (LZO), Antonie van Leeuwenhoeklaan 9, P.O. Box 1, Bilthoven, The Netherlands b Nutrition Sciences N.V., Vitamex Group, Belgium c Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
a r t i c l e
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Article history: Received 27 September 2010 Received in revised form 25 May 2011 Accepted 21 July 2011 Available online 29 July 2011 Keywords: Quantitative multiplex real-time PCR Bacillus anthracis Bacillus thuringiensis Anthrax DNA extraction efficiency PCR inhibitors
a b s t r a c t The DNA extraction efficiency from milk, whey, soy, corn gluten meal, wheat powders and heat-treated corn grain that were spiked with Bacillus anthracis and Bacillus thuringiensis spores was determined. Two steps were critical: lysis of the spores and binding of the free DNA to the DNA binding magnetic beads in the presence of the interfering powders. For the guanidine-thiocyanate based Nuclisens lysis buffer from Biomerieux we found that between 15 and 30% of the spores survived the lysis step. As most lysis buffers in DNA/RNA extraction kits are guanidine based it is likely that other lysis buffers will show a similar partial lysis of the Bacillus spores. Our results show that soybean flour and wheat flour inhibited the DNA extraction process strongest, leading to unreliable DNA extractions when using too much of the matrix. For corn gluten meal, heat-treated corn grain and milk powders, DNA extraction efficiencies in the presence of 100 mg and 10 mg of powder resulted in 70%–95% reduced DNA recoveries. The inhibition was, however, reliable and intermediate compared to the inhibition by soy and wheat. Whey powder had the lowest inhibitory effect on DNA-extraction efficiency and recoveries of 70–100% could be reached when using 10 mg of powder. The results show that reducing the amount of matrix leads to better DNA-extraction efficiencies, particularly for strongly inhibiting powders such as soy and wheat. Based on these results, a standard protocol to directly isolate DNA from micro-organisms present in complex matrixes such as food and feed powders was designed. © 2011 Elsevier B.V. All rights reserved.
1. Introduction For detection of contaminating micro-organisms in food and feed, PCR is becoming the method of choice (Malorny et al., 2003; Settanni and Corsetti, 2007). For food and feed samples, adding a (diluted) sample directly to a PCR reaction is for most cases not possible due to the presence of PCR inhibitors in the sample. For PCR based detection of such samples, one first needs to either enrich the culture prior to analysis or to extract the total DNA directly from the sample. Both methods have advantages and disadvantages. Culturing may give pure cultures which makes detection easy and makes DNA-extraction unnecessary. It has the disadvantage though of being time consuming, it is difficult to show a specific contamination when different types of micro-organisms are present, it is not generic in the sense that specific micro-organisms require specific growth conditions and quantification is difficult after enrichment. Direct extraction of total DNA has the advantage of being fast, generic and unbiased towards the DNA being
⁎ Corresponding author. Tel.: + 31 30 2747034; fax: + 31 30 2744434. E-mail address: [email protected] (P.R. Wielinga). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.07.023
extracted. The disadvantages include lack of sensitivity, the extraction of DNA from dead cells and inhibiting effects of the matrix from which the microbial DNA has to be extracted. Bacillus anthracis is part of the B. cereus family, consisting of the genetically closely related members: B. cereus, B. thuringiensis and B. anthracis (Bavykin et al., 2004). All three members form spores and are found world-wide. Their spores are highly resistant to different kinds of stress, enabling them to survive harsh environmental conditions (Driks, 2003), including some food processing procedures (Khan et al., 2009). B. anthracis is a zoonotic pathogen that may cause life threatening diseases in both animals and humans (Dixon et al., 1999; Mock and Fouet, 2001). After the anthrax letter attack in the US in 2001 (Jernigan et al., 2001), governments, the scientific community and the food and feed industries realised that in order to prevent a recurrence, development of simple and fast protocols for detecting highly dangerous pathogens (e.g. B. anthracis) is essential for an adequate and timely response to acts of bioterrorism. Since 2001, many different rapid methods have been developed and published for detection of DNA from biothreat agents such as B. anthracis, Francisella tularensis, Clostridium botulinum and Yersinia pestis (Antwerpen et al., 2008; Easterday et al., 2005; Fach
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et al., 2010; Hadjinicolaou et al., 2009; Skottman et al., 2007; Van Ert et al., 2007; Wielinga et al., 2010; Wilson et al., 2005). The essential step prior to this, DNA-extraction, however, has received less attention. There have been studies comparing different DNA extraction kits which focussed on using pure cultures or DNA. However, when extracting DNA directly from various matrices, the matrix can influence the efficiency of DNA extraction as well. The role of food and feed in a possible act of bioterrorism is a distinct possibility as shown by the use of infected food products in the past as a vector of intentional contamination (Anderson et al., 1996; Byrne et al., 2006; Hanson et al., 2005; Jernigan et al., 2001; Wein and Liu, 2005; see also the review by Keim and Wagner, 2009). Thus, contaminated products may need to be investigated. During such a bioterroristic event, PCR (involving extraction and detection) may be used to screen samples rapidly. Developing methodologies in preparation for such an event is important. In order to achieve this in a reliable manner, it is essential to understand the negative effects that different matrices may have on DNA-extraction, and take these into account when designing a suitable PCR detection protocol for these matrices. For the detection of a wide variety of so-called select agents these protocols need to be validated and applied under biosafety level 3 (BSL-3) or even higher laboratory safety conditions (Richmond and Nesby-O'Dell, 2002). This involves working under strict and controlled conditions, such as in an air tight pressure controlled glove box in a pressure controlled laboratory. This complicates the working procedures to a great extent and demands skilled and trained laboratory workers. It also demands well documented and validated working procedures which have been validated and are performed under the guidance of a biosafety officer. In previous studies, several different DNA-extraction kits were tested for DNA-extraction from different micro-organisms including Y. pestis, F. tularensis and B. anthracis (Coyne et al., 2004; Dauphin et al., 2009a,b; Miller et al., 1999; Ramisse et al., 1999; Sagova-Mareckova et al., 2008; Whitehouse and Hottel, 2007). Comparing six different DNA-extraction kits, the NucliSens kit was shown to have the most favourable characteristics (Dauphin et al., 2009a). We therefore chose to study this DNA-extraction kit further in our experiments. This kit uses a guanidine-thiocyanate solution which is a highly chaotropic chemical solution, and it is assumed that this type of medium results in complete lysis of all cell types, including spores. However, it has been shown that a certain fraction of spores can survive this harsh treatment (Miller et al., 1999; Dauphin and Bowen, 2009). This increases the challenge of extracting DNA from spores prior to PCR-detection. In this study, B. thuringiensis spores were initially used as a model organism for B. anthracis. Different food and feed powders were intentionally contaminated and the DNA-extraction efficiency of the NucliSens kit was studied under normal laboratory conditions. Furthermore, we determined to what extent PCR inhibitors were coextracted. Using the results from these experiments, we formulated an optimal protocol and tested this under BSL-3 conditions using 10 different food and feed powders spiked with B. anthracis spores. 2. Materials and methods 2.1. Food and feed samples Corn gluten meal, whey powder, wheat flour, soybean flour and heat-treated corn grain were supplied by Vitamex N.V. (Belgium). All these raw materials are used as major basic compounds for animal feed. The Irish milk and whey powders were from Dairygold Cooperative Ltd, Mitchelstown, Cork, Ireland. Low, medium and high heat skim milk powders (SMP) were manufactured by preheating milk to 72 °C × 12 s, 97 °C × 2 min and 120 °C × 2 min, respectively, prior to evaporation and drying. Typically, the powders contain 33% protein and 55% lactose. Whey powder was manufactured by evaporation and drying of cheese-whey. Typically, whey powder contains 12% protein
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and 75% lactose. Whey protein concentrate (WPC) was manufactured by ultrafiltration of whey to 35% protein prior to evaporation and drying of the retentate. Typically, WPC35 contains 35% protein and 55% lactose. 2.2. Polymerase chain reaction (PCR) Quantitative real time PCR (qPCR) reactions for B. anthracis and B. thuringiensis were conducted in multiplex format in 20 μl reaction mixtures using iQ Multiplex Powermix (Biorad) and primers and probes as described previously (Wielinga et al., 2010). The PCR was carried out under the following conditions: 5 min of denaturation at 95 °C followed by 50 cycles of 5 s 95 °C and 35 s 60 °C (amplification and detection) on an LC480 (Roche). The threshold cycle value (Ctvalue) at which a statistically significant increase of the fluorescence signal is first detected was determined by the second derivative method of the LightCycler 480 Software release 1.5.0. SP3 (Roche). 2.3. Strains and cultures The B. anthracis and B. cereus strains have been described previously (Carlin et al., 2006; Wahab et al., 2005). B. thuringiensis ATCC29730 spores were purchased from Raven Biological Laboratories (Omaha, Nebraska, USA). Vegetative cells were grown under aerobic conditions in brain hearth infusion (BHI) medium (Gibco) at 37 °C. Spores were generated by growing in sporulation medium. Sporulation was determined by comparing growth of untreated and heat treated (15 min 65 °C) cells on BHI agar plates and growing overnight at 30 °C. For B. cereus and B. thuringiensis, microscopic inspection confirmed that of the suspensions used more than 99% of the cells had sporulated. All handling of vegetative B. anthracis cells and spores was performed under BSL-3 conditions. 2.4. DNA extraction Cell lysis and DNA extractions were performed using NucliSens lysis buffer and NucliSens DNA Magnetic Extraction Reagents (Biomerieux, Boxtel, The Netherlands). To determine the DNA-extraction efficiency from spores in the presence of different powders under normal laboratory conditions, 100 mg powder was directly added to 10 mL of lysis buffer and spiked with 3 × 105 B. thuringiensis spores. After incubating and mixing (manually) for 10 min with magnetic silica particles the bead-bound DNA was recovered according to the manufacturer's instructions and the B. thuringiensis DNA-concentration was determined using a multiplex qPCR which detects both B. thuringiensis and B. anthracis DNA (Wielinga et al., 2010). To determine the extent of possible co-extracted PCR-inhibitors, the DNA extraction samples were spiked with B. anthracis DNA and compared to the measured Ct-values of non-powder extraction controls. Since diluting out possible inhibitors sometimes leads to a better PCRdetection we determined the Ct-values for both the undiluted and 10times diluted extracted DNA samples. For each sample the Ct-values were determined in duplicate. The percentage of DNA recovery was calculated relative to the control (set at 100%) using the comparative Ctmethod (Pfaffl, 2001; Livak, 2001) and a PCR efficiency of 2, using: Recovery (%) = 100%·[2 (Ct, sample − Ct, control)] −1. For each of the individual experiments the recovery was calculated, after which the average recovery was calculated by taking the mean of these values. Statistical significance was calculated using a Student's t-test; a value of p b 0.05 was considered significantly different from the control extraction. For B.anthracis, extractions were performed under BSL-3 conditions, adding 10 mg of powder to 10 mL of lysis buffer, spiking with 3 × 105 B. anthracis spores and extracting the DNA. Before removal from the isolator, all the DNA samples were filtered through a 0.22 μm Ultrafree Centrifugal Filter Unit (Millipore) to remove surviving spores.
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2.5. Survival of cells and spores in lysis buffer Spores and vegetative cells of either B. thuringiensis or B. cereus spores (10 6/mL) were incubated for 10 min in medium containing a decreasing percentage of lysis buffer. After incubation, 10-fold dilution series were made in BHI-medium supplemented with 0.05% TritonX100, the dilutions plated on BHI agar plates and grown overnight at 30 °C to determine the number of colony forming units (CFUs). 3. Results and discussion 3.1. Spore viability Spore viability was tested by determining the number of CFUs before and after incubation for 15 min at 65 °C. For both the B. cereus and B. thuringiensis spores, similar counts were obtained before and after 15 min heat treatment at 65 °C, showing a sporulation efficiency approaching 100%. Microscopic examination confirmed the sporulation efficiency of 99%–100%. Similar heat treatments of vegetative cells reduced the CFU count to zero. The efficiency of spore lysis by the NucliSens lysis buffer was assessed to determine if some spores survived this treatment. Fig. 1 shows the percentage of surviving B. cereus and B. thuringiensis spores and vegetative cells at decreasing lysis buffer concentrations. Treatment of vegetative cells with 100% lysis buffer led to a 100% reduction in CFUs. Treatment of B. cereus and B. thuringiensis spores with 100% lysis buffer led to 85% and 70% reduction in CFUs, respectively. This indicated that between 15% and 30% of the spores survived the lysis treatment and that their DNA was unavailable for extraction, which is important informa-
tion when PCR is being considered for screening purposes. In addition, this showed that although the sporulation level approached 100% and all spores appeared microscopically identical, there was a fraction of the spores withstanding the harsh treatment of the lysis buffer. 3.2. Direct DNA extraction from spores in the presence of 100 mg food and feed powders To determine the DNA extraction efficiency from spores in the presence of 10 different powders that are used in the food and feedindustry, 100 mg powder was spiked with 3 × 10 5 B. thuringiensis spores and total DNA was extracted. Table 1 shows the recovery (in %) of B. thuringiensis DNA and the PCR efficiency of the internal PCR control for ten different milk, whey, soy, wheat, corn and wheat powders, for both the undiluted and a 10-times diluted DNA extraction sample. Soybean flour and wheat flour inhibit the DNA-extraction process the most, followed by corn gluten meal, heat-treated corn grain and milk powders giving a relative intermediate inhibition. Whey powder was only mildly inhibitory. Comparing the undiluted and 10-times diluted samples showed that diluting the samples improved the sensitivity of B. thuringiensis DNA detection for several powders. For the undiluted samples only the whey powders showed a relatively good recovery (22% and 44%). Wheat flour and in particular the soybean flour showed very low recoveries. At a 10-fold dilution the recovery increased strongly for heat-treated corn grain, wheat, soy and corn gluten meal (see Table 1). For soy and corn gluten meal, this increase was also due to diluting out co-extracted PCR inhibitors, as the internal control detection efficiency went from 55% and 42% for the undiluted samples to about 100% for the 10-fold diluted sample (Table 1, compare fourth and fifth column). Further comparison of the internal control detection efficiencies showed that for the other the powders the PCR was not greatly affected by possible co-extracted substances. 3.3. Optimization of the direct DNA extraction protocol
Fig. 1. Percentage lysis of cells and spores in lysis buffer. After incubating vegetative cells (squares) and spores (diamonds) of B. thuringiensis (A) or B. cereus (B) for 10 min in the indicated percentage of lysis buffer, the colony forming units (CFU) were determined.
For both soybean flour and wheat flour, representing the two most interfering matrices, we optimised the extraction and determined the relationship between DNA recovery and several physical parameters. The relationship between DNA recovery and the amount of soybean flour and wheat flour added to 10 mL lysis buffer is shown in Fig. 2A. This shows that at concentrations above 10 mg powder per 10 mL lysis buffer, the DNA recovery was strongly inhibited and approaches 0%. At lower powder concentrations, the recovery increased exponentially and at 10 mg powder per 10 mL lysis buffer, the DNA could be recovered reproducibly from both wheat and soybean flour. Fig. 2B shows the relationship between DNA recovery and volume of lysis buffer used, while keeping the amount of soybean flour and incubation time constant. This shows that increasing the volume up to 50 mL led to a substantial decrease (almost 60%) in DNA recovery for the control sample (without powder added), which was due to dilution of the DNA. In contrast, and in spite of this decrease, recovery in the presence of 20 mg soybean flour was increased from almost 0% to 1.5%. This shows that decreasing the inhibition by lowering the powder concentration over-compensates the decreasing DNA concentration. Together, this counter intuitively shows that by lowering the amount of a contaminated powder in a DNA extraction one may increase ones chance of detecting the contamination, in particular when handling strongly inhibiting powders. Fig. 3 shows the relationship between DNA recovery and the incubation time in lysis buffer. For the no-powder control, maximum DNA binding occurred between 5 and 15 min. In the presence of wheat flour, recovery was maximal at 5 min and decreased over time. This indicates that, next to the DNA binding process other, as yet unknown, processes take place during lysis that make it disadvantageous to incubate for longer periods. For soybean flour, DNA recovery increased
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Table 1 The DNA recovery and the internal control detection efficiency for ten different powders (100 mg powder/10 mL lysis buffer) spiked with B. thuringiensis (3 × 105) spores. Sample
No addition control Corn gluten meal (Belgium) Whey powder (Belgium) Wheat flour (Belgium) Soybean flour (Belgium) Heat-treated corn grain (Belgium) SMP high heatc (Ireland) SMP medium heat (Ireland) SMP low heat (Ireland) WPC 35 UFd (Ireland) Whey powder (Ireland)
DNA recovery (%)
a
Internal control detection efficiency (%)
b
Undiluted
1 to 10
Undiluted
1 to 10
100% 6.3% ± 5.4% (29.2) 22% ± 14% (27.3) 0.7% ± 0.4% (37.5) 0.01% (37.0)e 3.7% ± 3.3% (30.4) 3.7% ± 0.2% (30.4) 4.2% ± 2.1% (30.1) 3.6% ± 2.9% (30.5) 5.1% ± 0.3% (29.6) 44% ± 15% (26.7)
100% 43% ± 72% (24.3) 60% ± 62% (24.1) 29% ± 45% (24.7) 2.1% ± 1.7% (29.6) 22% ± 27% (24.9) 4.0% ± 1.5% (27.8) 2.8% ± 0.4% (28.8) 2.6% ± 1.2% (29.0) 3.8% ± 1.7% (27.9) 38% ± 11% (24.4)
100% 90% ± 13% (29.1) 111% ± 16% (28.9) 121% ± 13% (28.9) 55% ± 50% (29.4) 42% ± 47% (29.5) 91% ± 7.0% (29.1) 117% ± 27% (28.9) 108% ± 19% (29.0) 149% ± 59% (28.8) 116% ± 29% (28.9)
100% 107% ± 14% (26.5) 112% ± 13% (26.4) 116% ± 25% (26.4) 103% ± 12% (26.5) 115% ± 11% (26.4) 105% ± 11% (26.5) 118% ± 8.0% (26.4) 119% ± 5.9% (26.4) 112% ± 24% (26.4) 107% ± 11% (26.5)
a For each extraction the Ct-value was determined in duplicate for the undiluted as well as a 10-times diluted sample. The DNA recovery (in %) was calculated relative to the control (set at 100%) using the comparative Ct-method (Pfaffl, 2001; Livak, 2001) using a PCR efficiency of 2: Recovery (%) = 100%·[2(Ct, sample − Ct, control)]−1. Data are given as the mean of n = 3 ± SD, except for the Irish powders which are given as the mean of n = 2 ± the average difference. Between brackets the average Ct-value is given for reference. The underlined values were not significantly different from the no powder extraction control calculated using a Student's t-test (p N 0.05). b As internal control, the DNA-extraction samples were spiked with B. anthracis DNA and detection efficiency calculated relative to the control (B. anthracis DNA alone). c Three different fractions of skim milk powders (SMP) were tested. d WPC — Whey Protein Concentrate 35% obtained by ultrafiltration. e For soybean flour two experiments resulted in a complete loss of PCR signal, while for the third experiment a weak curve with a Ct-value of 37 was observed.
over time and after 60 min the maximum was not reached, suggesting that longer incubation may increase the recovery even more. 3.4. Performance of DNA extraction method for B. anthracis spores under BSL-3 conditions From the data shown in Table 1 and Figs. 1–3 an optimised DNA extraction protocol that balanced the amount of powder, incubation time and volume of lysis buffer used, was formulated. The optimised protocol used 10 mL lysis buffer, 10 mg powder and 10 min lysis and DNA binding to the DNA-binding magnetic beads. This protocol was tested for the extraction of DNA from B. anthracis spores under BSL-3 conditions. Table 2 shows the recovery of B. anthracis from the powders tested (spiked with 3 × 10 5 B. anthracis spores). These data show that using this protocol, B. anthracis was reliably detected in all powders. Comparing the extraction efficiencies of the undiluted samples (Tables 1 and 2) showed that the 10-fold reduction of matrix increased the recovery for all powders tested, except for heat-treated corn grain. For soybean flour and wheat flour this gain was most evident. For these powders, the recovery went from 0.01% and 0.7% at 100 mg to 2% and 10% at 10 mg, respectively. For heat-treated corn grain, the recovery decreased from 3.7% to 0.5%, in contrast to what was expected. The reason for this is not clear and may be due to co-
Fig. 2. Effect of the amount of powder and the amount of lysis buffer on the DNA extraction efficiency. A) B. thuringiensis spores were incubated for 10 min at room temperature in 10 mL lysis buffer in the presence of the indicated amounts of soybean flour (diamonds) and wheat flour (squares) powder. Thereafter, the DNA was extracted and the amount determined by qPCR. The extraction efficiency was calculated relative to the control (no powder added) and is given as the average of n = 2 determinations (± the average difference). B) B. thuringiensis spores were incubated for 10 min with the indicated amount of lysis buffer in the presence (diamonds) and absence (squares) of 20 mg soybean flour. The extraction efficiency was calculated relative to the control (no powder added) determent with 10 mL lysis buffer.
Fig. 3. Effect of the incubation time on the DNA extraction efficiency. B. thuringiensis spores were incubated with soybean flour (squares) and wheat flour (triangles) for the indicated periods of time after which the DNA was extracted. The extraction efficiency was calculated and plotted relative to the amount of B. thuringiensis DNA extracted after 60 min for the no powder control (circles).
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Table 2 DNA extraction efficiencies under BSL-3 conditions. Powders (10 mg powder/10 mL lysis buffer) were spiked with 3 × 105 B. anthracis and incubated of 10 min in the presence of DNA binding beads after which DNA was isolated. Sample
No addition control Corn gluten meal (Belgium) Whey powder (Belgium) Wheat flour (Belgium) Soybean flour (Belgium) Heat-treated corn grain (Belgium) SMPb high heat (Ireland) SMP medium heat (Ireland) SMP low heat (Ireland) WPC 35 UFc (Ireland) Whey powder (Ireland)
B. anthracis spore DNA recovery (%)a Undiluted
1 to 10
100% 18% ± 12% (32.4) 70% ± 32% (31.2) 10% ± 7.7% (33.2) 2.0% ± 3.1% (37.0) 0.5% ± 1.1% (44.7) 34% ± 54% (31.7) 48% ± 80% (31.4) 17% ± 24% (32.5) 64% ± 52% (31.3) 100% ± 30% (31.0)
100% 25% ± 24% (34.7) 70% ± 43% (33.9) 13% ± 7.9% (35.5) 11% ± 11% (35.8) 16% ± 23% (35.2) 47% ± 77% (34.2) 58% ± 108% (34.0) 21% ± 37% (34.9) 67% ± 71% (33.9) 99% ± 64% (33.7)
a For each extraction the Ct-value was determined in duplicate for the undiluted as well as 10-times diluted sample. The extraction efficiencies were calculated as described in Table 1. Data are given as the mean of n = 3 ± SD. Between brackets the average Ct-value is given for reference. The underlined values were not significantly different from the no powder extraction control calculated using a Student's t-test (p N 0.05). b Three different fractions of skim milk powders (SMP) were tested. c WPC — Whey Protein Concentrate 35% obtained by ultrafiltration.
extracted B. anthracis PCR specific inhibitor. This is, however, not very likely as the two different organisms, B. thuringiensis and B. anthracis, for which the spores may react differently we assume it unlikely that their DNA reacts differently in these extractions. More relevant was the observation that in the lysis buffer suspension this powder was difficult to separate from the magnetic silica particles, which is particularly challenging under BSL-3 conditions. Diluting the extracted DNA before PCR detection to reduce the effects of co-extracted PCR inhibitors led to a strong gain in DNA recovery for heat-treated corn grain as well as for soybean flour. This indicated that a substantial amount of PCR inhibiting substance was still co-extracted from the 10 mg of these powders. For the other powders, diluting the DNA extract led to a small gain in the recovery, which indicated that at 10 mg no substantial amount of inhibiting substances was extracted from these powders. Our work shows that the detection method we present here can be satisfactorily applied in a BSL-3 laboratory for examination of food and feed powders potentially contaminated with B. anthracis. When using matrices other than the ones used here, it is advisable to test what effect these matrices have on DNA extraction before starting large scale laboratory work at BSL-3 or BSL-4 level. Testing a range between 100 and 5 mg may be good starting point for this; weighing less than 5 mg of material is complicated and may become unreliable under BSL-3 conditions. In the case of an emergency, for instance, when one assumes a bioterrorist attack, one needs to move swiftly. For such cases or when one is limited by the amount or knowledge of the sample material our optimised protocol may be used in a relatively reliable manner: use maximally 10 mg powder per 10 mL lysis buffer and determine the DNA concentration for both an undiluted and a 10-fold diluted extraction sample, preferentially using an internal extraction control. In the case of bulk volumes that need to be tested, one might scale up this amount while keeping the concentration of powder in the lysis buffer the same, or more practically when working under BSL-3 conditions, one may increase the sample size. 4. Concluding remarks We determined the spore DNA-extraction efficiency from ten different milk, whey, soy, corn and wheat powders spiked with B. anthracis and B. thuringiensis spores. We found that the critical steps in DNA-extractions are both lysis of the spores and binding of the DNA to the beads in the presence of the interfering powders. We showed
that between 15 and 30% of the spores survived the lysis step and thus their DNA was lost for detection. For our experiments, we used NucliSens lysis buffer, which is a guanidine thiocyanate based lysis buffer. Guanidine thiocyanate is a chemical which is generally used in many different lysis buffer formulations. Therefore, we think it is likely that for similar lysis buffers, in other DNA isolation kits, a similar partial lysis of the Bacillus spores may occur. We showed that (when using too much of the matrix) soybean flour and wheat flour inhibited the DNA-extraction process strongly, leading to unreliable DNA-extractions. For corn gluten meal, heattreated corn grain and milk powder the extractions at 100 mg and 10 mg powder resulted in an approximately 70%–95% reduced DNA recovery. Their inhibition was, however, reliable and intermediate when compared to the other powders tested. For whey powder, the DNA extractions were most reliable with DNA extraction efficiencies between 70 and 100% when using 10 mg powder and 20–40% when using 100 mg powder, respectively. The goal of our study was to report an optimised strategy for a general direct DNA extraction procedure for Bacillus spores in food and feed powders. Our experiments showed that for some strongly interfering powders, apparently logical approaches did not give the expected improved response. For such powders, adding more powder with a small trace of infectious agent did not lead to better, but to worse recoveries. From this, we conclude that when one is unfamiliar with the properties of a powder, it may be better to use less powder to be sure of extracting some DNA. In addition, our experiments showed that the incubation time of the powder in lysis buffer with the DNA-binding beads differs per powder and needs to be optimised. However, when one is unfamiliar with the exact matrix/powder, an incubation time of about 10–15 min seems best. Acknowledgement This work was supported by the European Union-funded Integrated Project BIOTRACER (contract #036272) under the 6th RTD Framework Programme of the European Union. References Anderson, R.M., Donnelly, C.A., Ferguson, N.M., Woolhouse, M.E., Watt, C.J., Udy, H.J., MaWhinney, S., Dunstan, S.P., Southwood, T.R., Wilesmith, J.W., Ryan, J.B., Hoinville, L.J., Hillerton, J.E., Austin, A.R., Wells, G.A., 1996. Transmission dynamics and epidemiology of BSE in British cattle. Nature 382, 779–788. Antwerpen, M.H., Zimmermann, P., Bewley, K., Frangoulidis, D., Meyer, H., 2008. Realtime PCR system targeting a chromosomal marker specific for Bacillus anthracis. Molecular and Cellular Probes 22, 313–315. Bavykin, S.G., Lysov, Y.P., Zakhariev, V., Kelly, J.J., Jackman, J., Stahl, D.A., Cherni, A., 2004. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms. Journal of Clinical Microbiology 42, 3711–3730. Byrne, B., Dunne, G., Bolton, D.J., 2006. Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll. Food Microbiology 23, 803–808. Carlin, F., Fricker, M., Pielaat, A., Heisterkamp, S., Shaheen, R., Salonen, M.S., Svensson, B., Nguyen-the, C., Ehling-Schulz, M., 2006. Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group. International Journal of Food Microbiology 109, 132–138. Coyne, S.R., Craw, P.D., Norwood, D.A., Ulrich, M.P., 2004. Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit. Journal of Clinical Microbiology 42, 4859–4862. Dauphin, L.A., Bowen, M.D., 2009. A simple method for the rapid removal of Bacillus anthracis spores from DNA preparations. Journal of Microbiological Methods 76, 212–214. Dauphin, L.A., Moser, B.D., Bowen, M.D., 2009a. 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. Journal of Microbiological Methods 76, 30–37. Dauphin, L.A., Stephens, K.W., Eufinger, S.C., Bowen, M.D., 2009b. Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples. Journal of Applied Microbiology 108, 163–172. Dixon, T.C., Meselson, M., Guillemin, J., Hanna, P.C., 1999. Anthrax. The New England Journal of Medicine 341, 815–826.
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