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A novel pentaplex real time (RT)- PCR high resolution melt curve assay for simultaneous detection of emetic and enterotoxin producing Bacillus cereus in food
Food Control 60 (2016) 560e568
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Food Control journal homepage: www.elsevier.com/locate/foodcont
A novel pentaplex real time (RT)- PCR high resolution melt curve assay for simultaneous detection of emetic and enterotoxin producing Bacillus cereus in food Fereidoun Forghani a, Prashant Singh b, Kun-Ho Seo c, Deog-Hwan Oh d, * a
Department of Microbiology, Immunology and Biochemistry, The University of Tennessee Health Science Center, Memphis, TN 38163, USA Department of Food Science & Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-1797, USA KU Center for Food Safety, College of Veterinary Medicine, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul, 143-701, Republic of Korea d Department of Bioconvergence Science and Technology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwon 200701, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 May 2015 Received in revised form 18 August 2015 Accepted 25 August 2015 Available online 28 August 2015
Bacillus cereus causing emetic and diarrheal type of food poisoning is widely distributed in nature and is therefore considered a major foodborne pathogen. There is a growing demand for fast, accurate, reliable and economic detection of potentially toxigenic B. cereus. To improve differential diagnosis of toxigenic B. cereus, a highly sensitive pentaplex RT- PCR high resolution melt curve assay was developed for simultaneous detection of 4 major enterotoxim genes (cytK, entFM, hblD, nheA) and emetic toxin gene (ces). The average melting temperatures (Tm) of PCR products were 72.2 C (ces), 74.23 C (cytK), 76.55 C (nheA), 78.42 C (entFM) and 81.90 (hblD). The multiplex assay was evaluated using 71 bacterial strains including 17 emetic B. cereus reference strains, 9 enterotoxic B. cereus reference strains, 4 B. cereus group members, 23 wild B. cereus strains, 18 non-target strains, and was further tested on artificially inoculated foods. The detection limit in food samples was approximately 103 CFU/g without enrichment and 101 CFU/g was observed following 7 h enrichment. The DNA intercalating dye SYTO9 used in this study generated high resolution melt curve peaks for the target strains and genes in which the peaks were sharp and easily distinguishable from each other. Thus, the developed multiplex real-time (RT) PCR approach can be a reliable tool for the identification of emetic and enterotoxic strains of B. cereus present in food and food-related samples. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Bacillus cereus Emetic strains Enterotoxic strains Multiplex real-time PCR Melt curve analysis
1. Introduction Bacillus species are ubiquitous and diverse both in the marine and terrestrial ecosystems (Oguntoyinbo, 2007). Accordingly, the “Bacillus cereus group” (B. cereus sensu lato) are Gram-positive, spore-forming, rod-shaped, opportunistic human pathogens and are ever-present threat in food (Abdou, Awny & Abozeid, 2011; €uber, Agata et al., 1994; Dierick et al., 2005; Fricker, Messelha Busch, Scherer, & Ehling-Schulz, 2007; Priha, Hallamaa, Saarela, & Raaska, 2004). B. cereus group comprises eight closely related species: B. cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus
* Corresponding author. No. 414, 309 #, Department of Bioconvergence Science and Technology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea. E-mail address: [email protected] (D.-H. Oh). http://dx.doi.org/10.1016/j.foodcont.2015.08.030 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
pseudomycoides, Bacillus anthracis, Bacillus weihenstephanensis, Bacillus cytotoxicus and Bacillus toyonensis, among which many of nez et al., them are of great medical and economic importance (Jime 2013). It is widely distributed in the environment and is a causative nez et al., agent of foodborne illness (Helgason et al., 2000; Jime 2013). Foodborne illness resulting from consumption of B. cereuscontaminated food may result in emetic or diarrheal type syndromes (Kim et al., 2010). Although B. cereus has been implicated in several foodborne outbreaks around the world, resulting in occasional hospitalization or even death (Dierick et al., 2005; Mahler et al., 1997), its true incidence is usually underestimated mainly due to similar symptoms to other types of food poisoning (Fricker et al., 2007). The diarrheal type of food poisoning is caused by heat-labile enterotoxins produced during vegetative growth of B. cereus in the small intestine (Ehling-Schulz, Fricker, & Scherer, 2004). The
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nonhemolytic enterotoxin (NHE), enterotoxin FM (EntFM), hemolysin BL (HBL) and cytotoxin K (CytK) are the main enterotoxins produced by B. cereus (Forghani, Kim, & Oh, 2014). Abdominal pain and diarrhea usually occur 8e16 h after ingestion of contaminated food and may be misdiagnosed with Clostridium perfringens food poisoning (Park et al., 2009). The NHE complex consists of three subunits which are encoded by nheA, nheB and nheC in one operon (Granum, O'Sullivan & Lund, 1999). Enterotoxin FM is a B. cereus cell wall peptidase implicated in virulence (Untergrasser et al., 2012). The HBL complex consists of three components L1, L2 and B which are encoded in one operon by the genes hblD, hblC and hblA, respectively, and has haemolytic and dermonecrotic activity (Corona, Fois, Mazzette, & De Santis, 2003), making it an important causative agent of the diarrheal syndrome (Beecher & Wong, 2000). Finally, the single-component protein toxin CytK is a b-barrel poreforming toxin with dermonecrotic, hemolytic and cytotoxic activities (Lund, DeBuyser, & Granum, 2000). The emetic syndrome is caused by emetic toxin (cereulide), a small cyclic peptide (D-O-LeuD-Ala-L-O-Val-L-Val3) belonging to dodecadepsipeptide molecules family. It is a heat-stable toxin causing nausea and vomiting approximately 1e5 h after consumption of contaminated food (Agata et al., 1994), with symptoms which resemble to Staphylococcus aureus food poisoning (Forghani, Kim & Oh, 2014). Conventional methods for the detection of B. cereus are time consuming, laborious and occasionally not precise. Thus, several molecular methods have been developed for the detection of B. cereus (Fern andez-No et al., 2011; Fricker, Reissbrodt, & Ehlingnchez, Garay, & Aznar, 2009). Schulz, 2008; Martínez-Blanch, Sa These tools either targeted a limited number of toxin producing genes or species-specific genes. However, assessment of multiple toxin genes which will help to reveal the full pathogenic potential of the strains by multiplex PCR assays might be better than only species detection for outbreak investigations (Ehling-schulz & €usser, 2013; Oh, Ham, & Cox, 2012). Messelha Several conventional PCR assays for the detection of B. cereus group as well as its emetic and/or enterotoxin producing strains have been developed (Ehling Schulz, Fricker, & Scherer, 2004; Ehling-Schulz et al., 2006; Forghani, Seo, & Oh, 2014; Ghelardi et al., 2002; Kim et al., 2012; Nakano et al., 2004). However, a major drawback of conventional PCR is the requirement for post-PCR analysis which is time consuming and bears the risk of falsepositive results due to laboratory contamination (Fan, Hamilton, Webster-Sesay, Nikolich, & Lindler, 2007; Fricker et al., 2007). On the other hand, real-time PCR allows sensitive high-throughput results with easy automation and does not require post-PCR detection procedures (Zhang et al., 2014). There are two main technologies applied in real-time PCR for the detection purpose. One is the application of fluorescent probes which will specifically attach to the target DNA sequence. The other procedure uses fluorescent dyes such as SYBR® Green I or SYTO9 intercalating with double-stranded (ds) DNA, binding to all amplicons generated in PCR reaction (Singh & Mustapha, 2014). The latter is less expensive, does not require probe design and PCR products can be differentiated by melting temperature (Tm) analysis which is also capable of multiplexing (Arya et al., 2005; Zhang et al., 2014). Thus far, some real-time assays for the detection of B. cereus group, enterotoxin producing strains, emetic toxin producing strains, quantification, viable cells detection and detection of B. cereus spores in food have been reported (Dzieciol, Fricker, Wagner, Hein, & Ehling-schulz, 2013; Fern andez-No et al., 2011; Martínez-Blanch et al., 2009, 2010, 2011). However, they either used dual-labeled probes such as TaqMan® which are expensive and require separate probe design and/or did not detect the toxin genes. Hence, the aim of this study was to develop a novel pentaplex RT- PCR high resolution melt curve assay for the wide
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range detection of emetic and enterotoxin producing B. cereus in food. 2. Materials and methods 2.1. Bacterial strains and culture conditions All 71 bacterial strains used in this study were obtained from the Dept. of Food Science and Biotechnology, Kangwon Natl. Univ. (Table 1). In brief, they consisted of 17 B. cereus emetic reference strains, 9 B. cereus enterotoxic reference strains, 4 members of B. cereus group, 23 wild B. cereus strains as well as 18 non-target strains. All strains were grown on nutrient agar (Difco, Detroit, Mich., USA) plates at 35 C for 24 h. A single colony was inoculated in LuriaeBertani broth (Difco) and incubated at 35 C for 12 h for further experiments. The emetic (F4810/72) and enterotoxic (ATCC 12480) B. cereus reference strains were used for the optimization and primary evaluation of the approach. 2.2. Bacterial DNA extraction For the DNA extraction, 1 ml of the overnight culture was centrifuged (15000 g; 2 min) and the pellet was resuspended in 100 ml of the PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA). The DNA was extracted according to the manufacturer's instructions. The concentration of DNA was adjusted to 10 ng/mL using a NanoDrop 2000 UV/VIS spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) at 260 nm and also its purity was checked. The DNA was stored at 20 C for the following experiments. 2.3. Primer design Table 2 shows the primers used in this study along with their design source and product size. Specific primers for the amplification of enterotoxin producing genes (cytK, entFM, hblD, nheA) and emetic toxin gene (ces) were designed using the Primer3 software (Untergrasser et al., 2012) and commercially synthesized using AccuOligo® technology (Bioneer, Daejeon, Korea) (http://eng. bioneer.com/products/Oligo/CustomOligonucleotides-technical. aspx). To further maximize the detection range of the primers, the International Union of Pure and Applied Chemistry (IUPAC) standards were taken into account if necessary. The primers were designed to keep the melting temperature (Tm) of the PCR amplicons between 70 C and 85 C and each amplicon Tm was separated by approximately 2 C. The specificity of the designed primers was tested using the Primer BLAST tool and the Tm of all amplicons was predicted using the BioEdit software (Hall, 1999). 2.4. Pentaplex RT- PCR high resolution melt curve assay In this study SYTO9-based 2 MeltDoctor™ HRM master mix (Applied Biosystems, Foster City, CA, USA) was selected over the SYBR® Green I-based master mix according to the literature reporting several advantages for SYTO9 dye in comparison with SYBR® Green. PCR amplification was performed in duplicate in a 20 ml reaction volume with 20 ng of DNA. The primer concentrations were 50, 150, 100, 50 and 100 nM for ces, cytK, nheA, entFM, and hblD, respectively. A StepOne™ real-time PCR (Applied Biosystems, Foster City, CA, USA) instrument with StepOne™ Software (Version 2.2.2; Applied Biosystems, Foster City, CA, USA) was used for the standardization of the real-time multiplex PCR assay. StepOne™ Software (Version 2.2.2; Applied Biosystems, Foster
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Table 1 Bacterial strains used in the study. Bacillus cereus emetic reference strains B. cereus F4810/72, JNHE 36, JNHE 80, KUGH 27, JNHE 82, KUGH 164, JNHE 7, JNHE 15, JNHE 60, JNHE 61, JNHE 23, JNHE 95, JNHE 13, JNHE 54, KNIH 20, KUGH 12, JNHE 88 Bacillus cereus enterotoxic reference strains B. cereus ATCC 12480, ATCC 27348, ATCC 11778, KCTC 1526, KCTC 1094, KCTC 1092, KCTC 1014, KCTC 1013, ATCC 13061 Bacillus cereus group members B. thuringiensis KCTC 1508, B. mycoides KCTC 3453, B. pseudomycoides KACC 12098, B. weihenstephanensis KACC 12001 Bacillus cereus wild strains B. cereus BC-64, BC-65, BC-66, BC-67, BC-68, BC-69, BC-70, BC-71, BC-72, BC-73, BC-74, BC-75, BC-76, BC-77, BC-78, BC-80, BC-81, 4303, 4300, 4165, 4158, 4154, 4153 Non-target strains Yersinia enterocolitica ATCC 23715, Campylobacter jejuni ATCC 33291, Vibrio parahaemolyticus ATCC 17802, Streptococcus pyogenes KCCM 11819, Micrococcus luteus KCCM 11211, Escherichia coli ATCC 3565, Pseudomonas putida KCCM 35479, Escherichia coli B0455, Escherichia coli B0499, Bacillus subtilis KCCM3135, Enterococcus faecalis ATCC 33186, Staphylococcus aureus ATCC 27729, Escherichia coli O157:H7 ATCC 43895, Streptococcus salivarius ATCC 19258, Listeria monocytogenes ATCC 19115, Escherichia coli KCCM 32396, Listeria monocytogenes ATCC 15313, Salmonella enteritidis ATCC 14028
Table 2 Primers used for multiplex real-time PCR high resolution melt curve assay. Target gene Sequence (50 / 30 ) ces cytK entFM hblD nheA
Product size (bp) Design source
GGG AGC CAA CAA CAA TGT CT 97 CAT GCA AAT GGT GTA ACG ATG TAA AGA AAC GRG CGC TGT TA 81 CTG GYG CTA GTG CAA CAT TA 255 GGA ACT GGA TAC GTA AGC CGG CCG TTA TGG TTA ATT T GCA TGG TCA ATT GGT GGT 163 CAC CAG CTG CTG TTC CTA TGA AAT TGT AAA TGC TGC A 127 TGT AAT TTG WGT CGC CTC TG
AY691650; DQ360825; DQ345790; JN222922; AY691650; DQ238109; DQ459072; JN112795; JN112796 AJ277962; DQ019311; JN222929; JN222928; JN222924; CP000001; AJ318875; AJ318877; AJ318876 AY789084; AY897207; EF453661; EF453653; EF453659 U63928; AJ007794; AY820178; AY820179; AY822582; AY822584 Y19005; DQ019312; DQ885236; DQ153261; DQ153257
City, CA, USA) was applied for the amplification and melt curve analysis. A two-step amplification protocol including initial denaturation at 95 C for 10 min followed by 40 cycles of 95 C for 15 s and 58 C for 1 min. A melt curve step was added at the end of the PCR amplification cycle. The high resolution melt curve analysis was performed from 60 C to 95 C, with gradual temperature increments of 0.1 ºC/s. A melt curve plot was prepared by plotting the negative derivative of fluorescence (Rn) versus temperature. The fluorescence signal was detected in the FAM channel of the realtime PCR instrument without quencher. Mean Tm value for each specific product was calculated by averaging the Tm values obtained after amplification in uniplex reactions. 2.5. Applicability and specificity of the assay The inclusivity and exclusivity of the enterotoxin genes targeting primers were evaluated using 9 enterotoxic B. cereus and 18 non-target strains. Primers designed to detect the emetic strains were tested using 17 emetic B. cereus and non-target strains. Also, four members of the B. cereus group were subjected to the multiplex real-time assay to test the ability in detection of toxigenic B. cereus group members. Finally, a panel of 23 wild B. cereus strains was included (Forghani, Kim & Oh, 2014) to confirm the approach applicability for detecting toxigenic B. cereus in food samples. Following specificity testing, a DNA suspension containing equal amounts of B. cereus ATCC 12480 enterotoxic and F4810/ 72 emetic reference strains DNA was prepared and subjected to multiplex RT- PCR to assure the ability of all 5 primers to work efficiently in a multiplex reaction in the presence of all five target genes. Furthermore, due to the strong background of food microflora that may obscure the accurate detection of the pathogen, B. cereus ATCC 12480 target DNA was mixed with the non-target DNA from Listeria monocytogenes (ATCC 19115) into the same reaction tube. High amount of the non-target DNA (20 ng) was added to the
tenfold serially diluted B. cereus DNA and its real-time PCR results were compared with a control standard dilution series containing only target DNA. 2.6. Sensitivity of the assay To detect the sensitivity of the developed pentaplex assay, DNA from B. cereus ATCC 12480 reference strain was isolated using PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. After stock DNA (10 ng/mL) was ten-fold serially diluted from 10 ng/ ml to 10 fg/ml of DNA, two microliters of each serially diluted DNA, in triplicate was used for the construction of standard curve. Microsoft Excel Software was used to compute regression coefficients and standard deviations for the standard curves. To further validate the sensitivity of the assay, overnight cultures of B. cereus ATCC 12480 (enterotoxic reference strain) and B. cereus F4810/72 were serially diluted in 0.1% buffered peptone water (BPW; pH 7.2) and enumerated using Tryptic Soy Agar (Becton Dickinson Diagnostic Systems, Sparks, MD, USA). DNA was similarly isolated from 1 ml of each dilution and subjected to RT- PCR by using 2 mL genomic DNA isolated from each dilution tube. 2.7. Analysis of artificially spiked food samples Infant formula, pasteurized milk, kimbab (Korean food containing rice and other ingredients rolled in seaweed), rice, pasta and tteok (Korean rice cake made with glutinous rice flour) were purchased from grocery stores in Chuncheon, Korea. These food products were tested for the absence of B. cereus by the standard reference culture methods (ISO 7932:2004; Fricker et al., 2007). Twenty five grams of each food sample was weighed in sterile stomacher bags (Nasco Whirl-Pak, Janesville, WI, USA), inoculated with B. cereus ATCC 12480 reference strain at different levels starting from 2.7 106 CFU/g to 2.7 101 CFU/g and was diluted in
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225 ml BPW (Becton Dickinson Diagnostic Systems, Sparks, MD, USA). Positive and negative process controls were also included as described elsewhere (Malorny et al., 2007). The negative control was prepared with sterilized enrichment media with the food sample that was enriched for 8 h. The positive control comprised of enrichment media, food and B. cereus ATCC 12480 at 2.7 101 CFU/ g sample. Enrichment of spiked food samples was performed in sterile stomacher bags (Nasco Whirl-Pak, Janesville, WI, USA) containing tryptic soy broth (TSB; Becton Dickinson Diagnostic Systems, Sparks, MD, USA) at 35 C with 150 rpm speed. The DNA from spiked food samples was isolated from the enriched broth withdrawn at intervals of 0, 3, 5 and 7 h. DNA extraction from food samples was performed using PrepMan® Ultra Sample Preparation Reagent and DNeasy® mericonTm Food Kit (Qiagen AB, Uppsala, Sweden) in parallel, according to the manufacturers' instructions. This extracted DNA (directly and also with 1:2 dilution) was used for RT- PCR (Singh & Mustapha, 2014). The reproducibility of the developed assay was tested using a panel of 10 B. cereus reference strains (5 enterotoxic, 5 emetic). 2.8. Statistical data analysis Reproducibility of the multiplex PCR was tested by determination of intra-assay (results of the same sample in different tubes in the same run) and inter-assay (results of the same sample in different runs) variation of crossing points and melting temperatures for a test panel of 10 B. cereus strains (5 emetic, 5 enterotoxic). The coefficient of variance (CV ¼ SD/arithmetic mean of Ct or Tm) was calculated for results of three replicates in the same run (intraassay) and for results of replicates in three separate runs (interassay). 3. Results
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PCR assay was high. Melt curves obtained for each reference strain were in agreement with the toxigenic profiles of the reference strains as previously reported (Forghani, Kim & Oh, 2014, Forghani, Seo, & Oh, 2014; Kim et al., 2010, 2012), confirming the specificity of the developed assay (Fig. 1A and B). All the B. cereus group members carrying toxin genes as previously reported were also detected (Fig. 1C and D). Finally, the assay successfully detected all the 23 wild B. cereus used in the study which were previously reported as enterotoxigenic strains (Forghani, Kim & Oh, 2014). In addition, when performing the assay on the DNA suspension containing equal amounts of DNA from B. cereus ATCC 12480 enterotoxic strain and F4810/72 emetic reference strain, 5 distinguishable peaks representing all the five toxin genes present in the reaction tube (ces, cytK, entFM, hblD, nheA) were produced (Fig. 2). Also, the presence of non-target L. monocytogenes did not change the performance of the assay (Fig. 3). 3.3. Sensitivity of the assay The developed pentaplex assay was found to be working efficiently over a DNA concentration range of 20 nge200 fg/reaction (Fig. 4). However, cytK and hblD primers showed a 10-fold higher sensitivity up to 20 fg of DNA per reaction. The obtained standard curve for each targeted gene with its respective regression coefficient (R2 value) is represented in Fig. 5. For further validation of the assay sensitivity, DNA isolated from decimally diluted broth cultures of B. cereus ATCC 12480 and B. cereus F4810/72 with a starting cell concentration of 1.6 106 CFU/mL and 3.3 106 CFU/mL, respectively. The results showed limit of detection (LOD) to be 1.6 102 CFU/mL and 3.3 102 CFU/mL for B. cereus ATCC 12480 and B. cereus F4810/72, respectively. All targets were successfully detected at this cell concentration.
3.1. Pentaplex RT- PCR high resolution melt (HRM) curve assay
3.4. Detection of B. cereus in artificially spiked food samples
The suitability of MeltDoctor™ HRM master mix for the multiplex detection of B. cereus emetic and enterotoxic strains was evaluated. Due to the efficiency of primers, use of saturating DNA binding SYTO9 dye in the MeltDoctor™ HRM master mix and application of slowest possible ramp rate (gradual temperature increments of 0.1 ºC/s), the melt curve peaks obtained for each target were equally sharp, regardless of their product size and GC content. The average melting temperatures (Tm) of all amplicons generated from the targeted genes in 5 reference strains are shown in Table 3.
The efficiency of the developed assay was further validated using 6 different food types. Table 4 shows the LOD of the method in food samples with or without enrichment. The developed multiplex assay was able to detect as low as 2.7 103 CFU/g in the food samples without an enrichment step using the DNeasy® mericonTm Food Kit. However, detection using PrepMan® Ultra Sample Preparation Reagent required 10-fold more organisms than in DNeasy for DNA extraction (Table 4). Following enrichment isolated DNA samples were diluted (1:2) using sterile nuclease-free distilled water. The RT- PCR assay could detect all of the target genes in the multiplex reaction with an LOD of 2.7 101 CFU/g with a 7 h enrichment period, regardless of the DNA extraction method used. Components of each food sample tend to affect the efficiency of DNA extraction/RT- PCR. Thus, samples with less complex matrices or lower amounts of fat or protein gave a lower Ct compared to more complex samples with the same level of contamination. The obtained Ct values of each primer in different foods in uniplex format are shown in Table 5. All foods were spiked at a level of 2.7 105 CFU/g B. cereus.
3.2. Applicability and specificity of the assay Assays were conducted to test the applicability and specificity of the multiplex RT- PCR HRM analysis using a concentration of 20 ng of DNA from microbial strains listed in Table 1. While all the target genes were amplified with Ct values in the range of 11e15, all the non-target strains were undetected (Ct values > 35) by the PCR machine, although 20 ng concentration of DNA employed in the
Table 3 Melting temperature (Tm) of amplicons in 8 B. cereus reference strains. Target gene
B. cereus strains
Mean Tm (ºC)
ces cytk nheA entFM hblD
B. B. B. B. B.
72.20 74.23 76.55 78.42 81.90
cereus cereus cereus cereus cereus
F4810/72, JNHE 36, JNHE 80, JNHE 60, JNHE 95 ATCC 12480, KCTC 1013, KCTC 1092, KCTC 1094, KCTC 1526 ATCC 12480, ATCC 13061, KCTC 1092, KCTC 1094, KCTC 1526 ATCC 12480, KCTC 1013, KCTC 1092, KCTC 1094, KCTC 1526 ATCC 12480, KCTC 1013, KCTC 1092, KCTC 1094, KCTC 1526
± ± ± ± ±
0.24 0.52 0.39 0.29 0.41
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Fig. 1. Melt curve peaks for the simultaneous detection of toxin genes in B. cereus reference strains: A) Detection of all 4 enterotoxin genes (cytK, entFM, hblD, nheA) of B. cereus ATCC 12480; B) Detection of all 4 enterotoxin genes (cytK, entFM, hblD, nheA) of B. cereus KCTC 1013; C) Detection of all 2 enterotoxin genes (cytK, entFM) of B. weinheistephanensis KACC 12001; D) Detection of entFM gene in B. pseudomycoides KACC 12098.
3.5. Reproducibility of the assay Results obtained from a panel of 10 B. cereus strains were used to assess intra-assay and inter-assay variation of the developed approach. Three replicates of each strain were applied in one run and three separate runs revealed average CV values for Tm of 0.025% and 0.06% and for Ct of 0.56% and 1.34%, respectively. 4. Discussion In recent years, melt curve-based RT- PCR assays have emerged as powerful tools for the detection of microbial pathogens ndez-No et al., 2011; Nam, Srinivasan, Gillespie, Murinda, & (Ferna Oliver, 2005; Newby, Hadfield, & Roberto, 2003; Price, Smith, Huygens, & Giffard, 2007), including B. cereus (Wehrle, Didier, €rtlbauer, 2010). In this study, we develMoravek, Dietrich, & Ma oped a novel multiplex RT- PCR high resolution melt curve assay for the simultaneous detection of emetic and enterotoxin producing B. cereus in food samples. The developed assay showed a sensitivity of 200 fg/reaction (25e30 genomic equivalents) for pure DNA, 103 CFU/g for spiked food without enrichment and a
sensitivity of approximately 101 CFU/g after a 7 h enrichment period for spiked food samples. The LODs obtained were enough for the analysis of B. cereus contaminated food without enrichment since contamination levels below 103 CFU/g are considered nchez, Garay, & Aznar, safe for consumers (Martínez-Blanch, Sa 2011). However, typical cell counts in food samples associated with food poisoning are usually much higher in the range of 105e108 CFU/g of B. cereus. The LOD obtained in this study was lower than those of some conventional PCR assays previously developed (Ehling-Schulz et al., 2004; Martínez-Blanch et al., 2011). Kim et al. (2012) and Zhang et al. (2014) also reported a LOD of 103 in different food matrices. Despite their advantages, all of the above mentioned methods need post-PCR procedures such as staining, gel electrophoresis and visualization which are not only time consuming and expensive but also may cause the risk for post-PCR contamination of the samples reducing the efficiency and simplicity of these conventional methods. On the other hand, RT- PCR allows high-throughput results with easy automation and does not require post-PCR detection procedures while it gives similar or higher sensitivity (Wehrle et al., 2010). Food samples artificially contaminated with B. cereus
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Fig. 2. Melt curve peaks for the simultaneous detection of all 4 enterotoxin genes (cytK, entFM, hblD, nheA) and emetic toxin gene (ces) in emetic/enterotoxic DNA suspension (equal amounts of DNA from B. cereus ATCC 12480 enterotoxic and F4810/72 emetic reference strain) by the pentaplex RT- PCR high resolution melt (HRM) curve assay.
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Fig. 4. Sensitivity of the multiplex assay in detecting B. cereus ATCC 12480 at DNA concentrations ranging from 20 ng to 200 fg using MeltDoctor™ HRM master mix.
concentrations lower than 3 log CFU/mL were not detected without enrichment, thus necessitating the addition of an enrichment step for lower concentrations. A LOD of approximately 101 CFU/g was achieved after an enrichment period of 7 h, which is similar to some
Fig. 3. Melt curve peaks of all 4 enterotoxin genes (cytK, entFM, hblD, nheA) of B. cereus ATCC 12480 detection using 20 ng of the target DNA with and without 20 ng of L. monocytogenes non-target DNA as background (A); Detection of all 4 enterotoxin genes of B. cereus ATCC 12480 using 200 fg of the target DNA with and without 20 ng of L. monocytogenes non-target DNA as background (B).
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Ct
28 26
ces
24
cytK
22
entFM
20 18
hblD
16
nheA
14 12 10 20 ng
2 ng
200 pg
20 pg
2 pg
200 fg
20 fg
DNA concentraƟon/reacƟon Fig. 5. Standard curve for all 5 target genes: ces (R2 ¼ 0.9937), cytK (R2 ¼ 0.9877), entFM (R2 ¼ 0.9772), hblD (R2 ¼ 0.9911) and nheA (R2 ¼ 0.9739) constructed using decimally diluted DNA.
previous reports (Forghani, Seo & Oh, 2014) and much shorter than the works of Wehrle et al. (2010) and Alarcon, Ohta, and Yokoyama (2005). Priha et al. (2004) reported a RT- PCR assay for the detection of B. cereus using 16SrDNA gene. Fricker et al. (2007) developed a RT-
PCR assay for the detection of emetic B. cereus using ces primer. nez-Blanch et al. (2009) developed a real-time PCR Whereas, Martıa assay for the detection and quantification of enterotoxigenic B. cereus which could not detect the emetic strains and its toxin nez-Blanch et al. (2010) detection range was limited. Later, Martıa developed a RT- PCR assay for detection and quantification of B. cereus group spores in food. Wehrle et al. (2010) developed a melt curve based RT- PCR approach for the detection of enteropathogenic B. cereus. However, the developed approach did not include entFM gene which is the most frequent toxin gene along with nhe in the majority of toxigenic B. cereus strains (Ngamwongsatit et al., 2008; Tewari, Singh, & Rashmi, 2013). More recently, Fern andeznez-Blanch et al. (2011) developed No et al. (2011) and Martıa real-time assays for the detection of B. cereus and viable B. cereus, respectively, without the ability to discriminate any of toxin genes. Finally, Dzieciol et al. (2013) developed a quantitative real-time approach for detection and differentiation of emetic (using ces gene) and non-emetic B. cereus. Each of the above mentioned approaches has its advantages but some of them do not detect any of the toxin genes responsible for causing B. cereus food poisoning. This is important due to the possible absence of toxin genes in some B. cereus strains has been nez et al., 2013; previously reported (Forghani, Seo & Oh, 2014; Jime Priha et al., 2004). Therefore, any detection approach without the ability of detecting toxin genes may detect non-pathogenic strains which are of no harm to the consumers. On the other hand, the
Table 4 Multiplex real-time PCR results of artificially contaminated food samples with/without enrichment using two different DNA extraction methods. Food sample
Spiked B. cereus concentration (CFU/g)
Real-time PCR result after enrichment DNeasy® mericonTm food kit
Infant formula
Milk
Kimbab
Rice
Pasta
Tteok
2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7
101 102 103 104 105 106 101 102 103 104 105 106 101 102 103 104 105 106 101 102 103 104 105 106 101 102 103 104 105 106 101 102 103 104 105 106
PrepMan® ultra sample preparation reagent
0h
3h
5h
7h
0h
3h
5h
7h
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
F. Forghani et al. / Food Control 60 (2016) 560e568
567
Table 5 Mean Ct values of each PCR primer in uniplex format, using different foods spiked with 2.7 105 CFU/g B. cereus. Target gene
Mean Ct value Rice
ces cytK entFM hblD nheA
17.52 15.74 19.21 18.38 18.64
Tteok ± ± ± ± ±
0.11 0.09 0.07 0.08 0.18
18.13 16.11 19.27 18.55 18.97
Pasta ± ± ± ± ±
0.09 0.09 0.27 0.22 0.17
18.32 16.29 19.46 18.64 19.34
± ± ± ± ±
0.26 0.17 0.24 0.28 0.16
Infant formula
Milk
± ± ± ± ±
19.15 16.81 19.95 19.52 20.29
18.76 16.58 19.63 18.97 19.69
0.83 0.46 0.39 0.31 0.38
Kimbab ± ± ± ± ±
0.76 0.22 0.14 0.33 0.25
19.47 17.93 20.88 20.47 21.16
± ± ± ± ±
0.38 0.61 0.51 0.39 0.22
Values are means of 3 measurements ± SD.
other reported methods lacked the ability to detect one or more of the major enterotoxins (cytK, entFM, hblD, nheA) or emetic toxin (ces). Developing detection methods for various types of toxins from enteropathogenic B. cereus in food samples are very important as there is a high variation in the distribution of toxin genes among B. cereus strains and several toxigenic profiles are available (Forghani, Kim & Oh, 2014). Also, considering the gene polymorphism which may occur in the toxin genes of wild B. cereus strains, a higher number of multiplexing experimental designs will strongly increase the detection rate in case of probable primer mismatch for some of the target genes (Schoeni & Wong, 1999; Thaenthanee, Wong, & Panbangred, 2005). Therefore, the present approach was developed to simultaneously detect all the four major enterotoxin genes as well as the emetic toxin genes in a single reaction. Food samples contain many organic and inorganic compounds, such as enzymes, polysaccharides, proteins and salts (Forghani, Seo , & Skapova, & Oh, 2014; Spanov a, Rittich, Karpískov a, Cechov a 2000). However, none of these inhibitors can be detected using the routine spectrophotometric measurements of DNA samples. This explains the reduction in LOD along with complexity of the food matrix as well as shorter enrichment time for foods with simpler matrix. Thus, application of an appropriate DNA extraction method capable of maximum recovery and inhibitor removal is a critical point for achieving high sensitivity of the method. As expected, this study showed a ten times higher sensitivity for the realtime approach using a specific (DNeasy® mericonTm) DNA extraction kit compared to PrepMan® Ultra Sample Preparation Reagent (Table 4). However, the latter has the advantage of shorter DNA extraction time less than 20 min compared to approximately 2 h needed for DNeasy® kit, making it the best choice for DNA isolation from bacterial cultures or food samples with higher bacterial loads (e.g. longer enrichment times). Other measures that could reduce the level of PCR inhibitors include a low speed centrifugation step to sediment large solid compound in the food homogenate upper liquid part (Singh & Mustapha, 2014) which we added to the routine DNA extraction protocols for both kits in this study. Probe-based methods are usually more specific than melt curve real-time PCR assays, but the specificity of the melt curve assays can also be maximized via appropriate primer design. Furthermore, melt curve based assays can be more sensitive (Fricker et al., 2007) and have a much lower running cost (Singh & Mustapha, 2014). As the probe itself has a limited storage life compared to the primers and master mix, it is also difficult to optimize for high multiplex numbers (such as our pentaplex approach) due to real-time chemistry as well as the necessity of multiple detection channels in the real-time PCR instrument. Thus, intercalating dyes such as SYTO9 have become the preferred choice and are being increasingly used for bacterial identification (Eischeid, 2011; Tong & Giffard, 2012). In this study, SYTO9 was selected over SYBR® Green I due to its several advantages. SYBR® Green I has a number of limitations that include the inhibition of PCR, preferential binding to GC-rich sequences and more noise production during the melt curve
analysis (Dragan et al., 2012; Giglio, Monis, & Saint, 2003; Karsai, Müller, Platz & Hauser, 2002; Nath, Sarosy, Hahn, & Di Como, 2000; Reed, Kent, & Wittwer, 2007). On the other hand, SYTO9 being a saturating DNA binding dye does not inhibit PCR and also does not show preferential PCR amplicon binding making it a better choice for multiplexing method (Monis, Giglio, & Saint, 2005). Most importantly, it provides the possibility of high resolution melt curve analysis which was of great importance in this work because of the close Tm of 5 different amplicons present in the same reaction tube (amplicon separated by approximately 2 C difference from each other) and the ability of SYTO9 to distinguish between amplicons with close Tm. However, in order to maximize the assay resolution, the default 0.3 C/s ramp rate (melting step) of StepOne™ real-time PCR machine was manually changed to 0.1 C/s resulting in much slower amplicon melting and generation of data with much higher resolution. In conclusion, although traditional culture based methods are still the gold standard for the detection of B. cereus, they require about 3 days (Martínez-Blanch et al., 2009) in food for confirming the presence of B. cereus. The current assay revealed a detection limit of 103 CFU/g or higher in less than 6 h, but with an enrichment step of 7 h it could detect contamination levels as low as 101 CFU/g food and does not require time-consuming and contaminationprone post-PCR steps. It provides additional information on the toxigenic profiles of the detected strains. This advantage makes the present approach a candidate biomarker for other applications such as in clinical settings. The pentaplex RT- PCR high resolution melt curve assay described in this study offers a simple workflow with a total run time of less than 12 h, with such a wide detection range of both emetic and enterotoxic strains of B. cereus. It is an economical alternative to the previous methods both for the melt curve analysis technology instead of the probe-based technology for real-time detection and the high number of multiplexing covering 5 different toxin genes in a single reaction. Acknowledgments This research was supported by Bio-industry Technology Development Program (112137-3) Ministry of Agriculture, Food and Rural Affairs. References Abdou, M. A., Awny, N. M., & Abozeid, A. A. E. M. (2011). Prevalence of toxicogenic bacteria in some foods and detection of Bacillus cereus and Staphylococcus aureus enterotoxin genes using multiplex PCR. Annals of Microbiology, 62(2), 569e580. Agata, N., Mori, M., Ohta, M., Suwan, S., Ohtani, I., & Isobe, M. (1994). A novel dodecadepsipeptide, cereulide, isolated from Bacillus cereus vacuole formation in Hep-2 cells. FEMS Microbiology Letters, 121, 31e34. Alarcon, B., Ohta, M., & Yokoyama, K. (2005). PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food. Journal of Applied Microbiology, 100, 352e364. Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N., & Patel, H. R. (2005). Basic principles of real-time quantitative PCR. Expert Review of Molecular Diagnostics, 5, 209e219.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
A novel pentaplex real time (RT)- PCR high resolution melt curve assay for simultaneous detection of emetic and enterotoxin producing Bacillus cereus in food Fereidoun Forghani a, Prashant Singh b, Kun-Ho Seo c, Deog-Hwan Oh d, * a
Department of Microbiology, Immunology and Biochemistry, The University of Tennessee Health Science Center, Memphis, TN 38163, USA Department of Food Science & Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-1797, USA KU Center for Food Safety, College of Veterinary Medicine, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul, 143-701, Republic of Korea d Department of Bioconvergence Science and Technology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwon 200701, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 May 2015 Received in revised form 18 August 2015 Accepted 25 August 2015 Available online 28 August 2015
Bacillus cereus causing emetic and diarrheal type of food poisoning is widely distributed in nature and is therefore considered a major foodborne pathogen. There is a growing demand for fast, accurate, reliable and economic detection of potentially toxigenic B. cereus. To improve differential diagnosis of toxigenic B. cereus, a highly sensitive pentaplex RT- PCR high resolution melt curve assay was developed for simultaneous detection of 4 major enterotoxim genes (cytK, entFM, hblD, nheA) and emetic toxin gene (ces). The average melting temperatures (Tm) of PCR products were 72.2 C (ces), 74.23 C (cytK), 76.55 C (nheA), 78.42 C (entFM) and 81.90 (hblD). The multiplex assay was evaluated using 71 bacterial strains including 17 emetic B. cereus reference strains, 9 enterotoxic B. cereus reference strains, 4 B. cereus group members, 23 wild B. cereus strains, 18 non-target strains, and was further tested on artificially inoculated foods. The detection limit in food samples was approximately 103 CFU/g without enrichment and 101 CFU/g was observed following 7 h enrichment. The DNA intercalating dye SYTO9 used in this study generated high resolution melt curve peaks for the target strains and genes in which the peaks were sharp and easily distinguishable from each other. Thus, the developed multiplex real-time (RT) PCR approach can be a reliable tool for the identification of emetic and enterotoxic strains of B. cereus present in food and food-related samples. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Bacillus cereus Emetic strains Enterotoxic strains Multiplex real-time PCR Melt curve analysis
1. Introduction Bacillus species are ubiquitous and diverse both in the marine and terrestrial ecosystems (Oguntoyinbo, 2007). Accordingly, the “Bacillus cereus group” (B. cereus sensu lato) are Gram-positive, spore-forming, rod-shaped, opportunistic human pathogens and are ever-present threat in food (Abdou, Awny & Abozeid, 2011; €uber, Agata et al., 1994; Dierick et al., 2005; Fricker, Messelha Busch, Scherer, & Ehling-Schulz, 2007; Priha, Hallamaa, Saarela, & Raaska, 2004). B. cereus group comprises eight closely related species: B. cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus
* Corresponding author. No. 414, 309 #, Department of Bioconvergence Science and Technology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea. E-mail address: [email protected] (D.-H. Oh). http://dx.doi.org/10.1016/j.foodcont.2015.08.030 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
pseudomycoides, Bacillus anthracis, Bacillus weihenstephanensis, Bacillus cytotoxicus and Bacillus toyonensis, among which many of nez et al., them are of great medical and economic importance (Jime 2013). It is widely distributed in the environment and is a causative nez et al., agent of foodborne illness (Helgason et al., 2000; Jime 2013). Foodborne illness resulting from consumption of B. cereuscontaminated food may result in emetic or diarrheal type syndromes (Kim et al., 2010). Although B. cereus has been implicated in several foodborne outbreaks around the world, resulting in occasional hospitalization or even death (Dierick et al., 2005; Mahler et al., 1997), its true incidence is usually underestimated mainly due to similar symptoms to other types of food poisoning (Fricker et al., 2007). The diarrheal type of food poisoning is caused by heat-labile enterotoxins produced during vegetative growth of B. cereus in the small intestine (Ehling-Schulz, Fricker, & Scherer, 2004). The
F. Forghani et al. / Food Control 60 (2016) 560e568
nonhemolytic enterotoxin (NHE), enterotoxin FM (EntFM), hemolysin BL (HBL) and cytotoxin K (CytK) are the main enterotoxins produced by B. cereus (Forghani, Kim, & Oh, 2014). Abdominal pain and diarrhea usually occur 8e16 h after ingestion of contaminated food and may be misdiagnosed with Clostridium perfringens food poisoning (Park et al., 2009). The NHE complex consists of three subunits which are encoded by nheA, nheB and nheC in one operon (Granum, O'Sullivan & Lund, 1999). Enterotoxin FM is a B. cereus cell wall peptidase implicated in virulence (Untergrasser et al., 2012). The HBL complex consists of three components L1, L2 and B which are encoded in one operon by the genes hblD, hblC and hblA, respectively, and has haemolytic and dermonecrotic activity (Corona, Fois, Mazzette, & De Santis, 2003), making it an important causative agent of the diarrheal syndrome (Beecher & Wong, 2000). Finally, the single-component protein toxin CytK is a b-barrel poreforming toxin with dermonecrotic, hemolytic and cytotoxic activities (Lund, DeBuyser, & Granum, 2000). The emetic syndrome is caused by emetic toxin (cereulide), a small cyclic peptide (D-O-LeuD-Ala-L-O-Val-L-Val3) belonging to dodecadepsipeptide molecules family. It is a heat-stable toxin causing nausea and vomiting approximately 1e5 h after consumption of contaminated food (Agata et al., 1994), with symptoms which resemble to Staphylococcus aureus food poisoning (Forghani, Kim & Oh, 2014). Conventional methods for the detection of B. cereus are time consuming, laborious and occasionally not precise. Thus, several molecular methods have been developed for the detection of B. cereus (Fern andez-No et al., 2011; Fricker, Reissbrodt, & Ehlingnchez, Garay, & Aznar, 2009). Schulz, 2008; Martínez-Blanch, Sa These tools either targeted a limited number of toxin producing genes or species-specific genes. However, assessment of multiple toxin genes which will help to reveal the full pathogenic potential of the strains by multiplex PCR assays might be better than only species detection for outbreak investigations (Ehling-schulz & €usser, 2013; Oh, Ham, & Cox, 2012). Messelha Several conventional PCR assays for the detection of B. cereus group as well as its emetic and/or enterotoxin producing strains have been developed (Ehling Schulz, Fricker, & Scherer, 2004; Ehling-Schulz et al., 2006; Forghani, Seo, & Oh, 2014; Ghelardi et al., 2002; Kim et al., 2012; Nakano et al., 2004). However, a major drawback of conventional PCR is the requirement for post-PCR analysis which is time consuming and bears the risk of falsepositive results due to laboratory contamination (Fan, Hamilton, Webster-Sesay, Nikolich, & Lindler, 2007; Fricker et al., 2007). On the other hand, real-time PCR allows sensitive high-throughput results with easy automation and does not require post-PCR detection procedures (Zhang et al., 2014). There are two main technologies applied in real-time PCR for the detection purpose. One is the application of fluorescent probes which will specifically attach to the target DNA sequence. The other procedure uses fluorescent dyes such as SYBR® Green I or SYTO9 intercalating with double-stranded (ds) DNA, binding to all amplicons generated in PCR reaction (Singh & Mustapha, 2014). The latter is less expensive, does not require probe design and PCR products can be differentiated by melting temperature (Tm) analysis which is also capable of multiplexing (Arya et al., 2005; Zhang et al., 2014). Thus far, some real-time assays for the detection of B. cereus group, enterotoxin producing strains, emetic toxin producing strains, quantification, viable cells detection and detection of B. cereus spores in food have been reported (Dzieciol, Fricker, Wagner, Hein, & Ehling-schulz, 2013; Fern andez-No et al., 2011; Martínez-Blanch et al., 2009, 2010, 2011). However, they either used dual-labeled probes such as TaqMan® which are expensive and require separate probe design and/or did not detect the toxin genes. Hence, the aim of this study was to develop a novel pentaplex RT- PCR high resolution melt curve assay for the wide
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range detection of emetic and enterotoxin producing B. cereus in food. 2. Materials and methods 2.1. Bacterial strains and culture conditions All 71 bacterial strains used in this study were obtained from the Dept. of Food Science and Biotechnology, Kangwon Natl. Univ. (Table 1). In brief, they consisted of 17 B. cereus emetic reference strains, 9 B. cereus enterotoxic reference strains, 4 members of B. cereus group, 23 wild B. cereus strains as well as 18 non-target strains. All strains were grown on nutrient agar (Difco, Detroit, Mich., USA) plates at 35 C for 24 h. A single colony was inoculated in LuriaeBertani broth (Difco) and incubated at 35 C for 12 h for further experiments. The emetic (F4810/72) and enterotoxic (ATCC 12480) B. cereus reference strains were used for the optimization and primary evaluation of the approach. 2.2. Bacterial DNA extraction For the DNA extraction, 1 ml of the overnight culture was centrifuged (15000 g; 2 min) and the pellet was resuspended in 100 ml of the PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA). The DNA was extracted according to the manufacturer's instructions. The concentration of DNA was adjusted to 10 ng/mL using a NanoDrop 2000 UV/VIS spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) at 260 nm and also its purity was checked. The DNA was stored at 20 C for the following experiments. 2.3. Primer design Table 2 shows the primers used in this study along with their design source and product size. Specific primers for the amplification of enterotoxin producing genes (cytK, entFM, hblD, nheA) and emetic toxin gene (ces) were designed using the Primer3 software (Untergrasser et al., 2012) and commercially synthesized using AccuOligo® technology (Bioneer, Daejeon, Korea) (http://eng. bioneer.com/products/Oligo/CustomOligonucleotides-technical. aspx). To further maximize the detection range of the primers, the International Union of Pure and Applied Chemistry (IUPAC) standards were taken into account if necessary. The primers were designed to keep the melting temperature (Tm) of the PCR amplicons between 70 C and 85 C and each amplicon Tm was separated by approximately 2 C. The specificity of the designed primers was tested using the Primer BLAST tool and the Tm of all amplicons was predicted using the BioEdit software (Hall, 1999). 2.4. Pentaplex RT- PCR high resolution melt curve assay In this study SYTO9-based 2 MeltDoctor™ HRM master mix (Applied Biosystems, Foster City, CA, USA) was selected over the SYBR® Green I-based master mix according to the literature reporting several advantages for SYTO9 dye in comparison with SYBR® Green. PCR amplification was performed in duplicate in a 20 ml reaction volume with 20 ng of DNA. The primer concentrations were 50, 150, 100, 50 and 100 nM for ces, cytK, nheA, entFM, and hblD, respectively. A StepOne™ real-time PCR (Applied Biosystems, Foster City, CA, USA) instrument with StepOne™ Software (Version 2.2.2; Applied Biosystems, Foster City, CA, USA) was used for the standardization of the real-time multiplex PCR assay. StepOne™ Software (Version 2.2.2; Applied Biosystems, Foster
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Table 1 Bacterial strains used in the study. Bacillus cereus emetic reference strains B. cereus F4810/72, JNHE 36, JNHE 80, KUGH 27, JNHE 82, KUGH 164, JNHE 7, JNHE 15, JNHE 60, JNHE 61, JNHE 23, JNHE 95, JNHE 13, JNHE 54, KNIH 20, KUGH 12, JNHE 88 Bacillus cereus enterotoxic reference strains B. cereus ATCC 12480, ATCC 27348, ATCC 11778, KCTC 1526, KCTC 1094, KCTC 1092, KCTC 1014, KCTC 1013, ATCC 13061 Bacillus cereus group members B. thuringiensis KCTC 1508, B. mycoides KCTC 3453, B. pseudomycoides KACC 12098, B. weihenstephanensis KACC 12001 Bacillus cereus wild strains B. cereus BC-64, BC-65, BC-66, BC-67, BC-68, BC-69, BC-70, BC-71, BC-72, BC-73, BC-74, BC-75, BC-76, BC-77, BC-78, BC-80, BC-81, 4303, 4300, 4165, 4158, 4154, 4153 Non-target strains Yersinia enterocolitica ATCC 23715, Campylobacter jejuni ATCC 33291, Vibrio parahaemolyticus ATCC 17802, Streptococcus pyogenes KCCM 11819, Micrococcus luteus KCCM 11211, Escherichia coli ATCC 3565, Pseudomonas putida KCCM 35479, Escherichia coli B0455, Escherichia coli B0499, Bacillus subtilis KCCM3135, Enterococcus faecalis ATCC 33186, Staphylococcus aureus ATCC 27729, Escherichia coli O157:H7 ATCC 43895, Streptococcus salivarius ATCC 19258, Listeria monocytogenes ATCC 19115, Escherichia coli KCCM 32396, Listeria monocytogenes ATCC 15313, Salmonella enteritidis ATCC 14028
Table 2 Primers used for multiplex real-time PCR high resolution melt curve assay. Target gene Sequence (50 / 30 ) ces cytK entFM hblD nheA
Product size (bp) Design source
GGG AGC CAA CAA CAA TGT CT 97 CAT GCA AAT GGT GTA ACG ATG TAA AGA AAC GRG CGC TGT TA 81 CTG GYG CTA GTG CAA CAT TA 255 GGA ACT GGA TAC GTA AGC CGG CCG TTA TGG TTA ATT T GCA TGG TCA ATT GGT GGT 163 CAC CAG CTG CTG TTC CTA TGA AAT TGT AAA TGC TGC A 127 TGT AAT TTG WGT CGC CTC TG
AY691650; DQ360825; DQ345790; JN222922; AY691650; DQ238109; DQ459072; JN112795; JN112796 AJ277962; DQ019311; JN222929; JN222928; JN222924; CP000001; AJ318875; AJ318877; AJ318876 AY789084; AY897207; EF453661; EF453653; EF453659 U63928; AJ007794; AY820178; AY820179; AY822582; AY822584 Y19005; DQ019312; DQ885236; DQ153261; DQ153257
City, CA, USA) was applied for the amplification and melt curve analysis. A two-step amplification protocol including initial denaturation at 95 C for 10 min followed by 40 cycles of 95 C for 15 s and 58 C for 1 min. A melt curve step was added at the end of the PCR amplification cycle. The high resolution melt curve analysis was performed from 60 C to 95 C, with gradual temperature increments of 0.1 ºC/s. A melt curve plot was prepared by plotting the negative derivative of fluorescence (Rn) versus temperature. The fluorescence signal was detected in the FAM channel of the realtime PCR instrument without quencher. Mean Tm value for each specific product was calculated by averaging the Tm values obtained after amplification in uniplex reactions. 2.5. Applicability and specificity of the assay The inclusivity and exclusivity of the enterotoxin genes targeting primers were evaluated using 9 enterotoxic B. cereus and 18 non-target strains. Primers designed to detect the emetic strains were tested using 17 emetic B. cereus and non-target strains. Also, four members of the B. cereus group were subjected to the multiplex real-time assay to test the ability in detection of toxigenic B. cereus group members. Finally, a panel of 23 wild B. cereus strains was included (Forghani, Kim & Oh, 2014) to confirm the approach applicability for detecting toxigenic B. cereus in food samples. Following specificity testing, a DNA suspension containing equal amounts of B. cereus ATCC 12480 enterotoxic and F4810/ 72 emetic reference strains DNA was prepared and subjected to multiplex RT- PCR to assure the ability of all 5 primers to work efficiently in a multiplex reaction in the presence of all five target genes. Furthermore, due to the strong background of food microflora that may obscure the accurate detection of the pathogen, B. cereus ATCC 12480 target DNA was mixed with the non-target DNA from Listeria monocytogenes (ATCC 19115) into the same reaction tube. High amount of the non-target DNA (20 ng) was added to the
tenfold serially diluted B. cereus DNA and its real-time PCR results were compared with a control standard dilution series containing only target DNA. 2.6. Sensitivity of the assay To detect the sensitivity of the developed pentaplex assay, DNA from B. cereus ATCC 12480 reference strain was isolated using PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. After stock DNA (10 ng/mL) was ten-fold serially diluted from 10 ng/ ml to 10 fg/ml of DNA, two microliters of each serially diluted DNA, in triplicate was used for the construction of standard curve. Microsoft Excel Software was used to compute regression coefficients and standard deviations for the standard curves. To further validate the sensitivity of the assay, overnight cultures of B. cereus ATCC 12480 (enterotoxic reference strain) and B. cereus F4810/72 were serially diluted in 0.1% buffered peptone water (BPW; pH 7.2) and enumerated using Tryptic Soy Agar (Becton Dickinson Diagnostic Systems, Sparks, MD, USA). DNA was similarly isolated from 1 ml of each dilution and subjected to RT- PCR by using 2 mL genomic DNA isolated from each dilution tube. 2.7. Analysis of artificially spiked food samples Infant formula, pasteurized milk, kimbab (Korean food containing rice and other ingredients rolled in seaweed), rice, pasta and tteok (Korean rice cake made with glutinous rice flour) were purchased from grocery stores in Chuncheon, Korea. These food products were tested for the absence of B. cereus by the standard reference culture methods (ISO 7932:2004; Fricker et al., 2007). Twenty five grams of each food sample was weighed in sterile stomacher bags (Nasco Whirl-Pak, Janesville, WI, USA), inoculated with B. cereus ATCC 12480 reference strain at different levels starting from 2.7 106 CFU/g to 2.7 101 CFU/g and was diluted in
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225 ml BPW (Becton Dickinson Diagnostic Systems, Sparks, MD, USA). Positive and negative process controls were also included as described elsewhere (Malorny et al., 2007). The negative control was prepared with sterilized enrichment media with the food sample that was enriched for 8 h. The positive control comprised of enrichment media, food and B. cereus ATCC 12480 at 2.7 101 CFU/ g sample. Enrichment of spiked food samples was performed in sterile stomacher bags (Nasco Whirl-Pak, Janesville, WI, USA) containing tryptic soy broth (TSB; Becton Dickinson Diagnostic Systems, Sparks, MD, USA) at 35 C with 150 rpm speed. The DNA from spiked food samples was isolated from the enriched broth withdrawn at intervals of 0, 3, 5 and 7 h. DNA extraction from food samples was performed using PrepMan® Ultra Sample Preparation Reagent and DNeasy® mericonTm Food Kit (Qiagen AB, Uppsala, Sweden) in parallel, according to the manufacturers' instructions. This extracted DNA (directly and also with 1:2 dilution) was used for RT- PCR (Singh & Mustapha, 2014). The reproducibility of the developed assay was tested using a panel of 10 B. cereus reference strains (5 enterotoxic, 5 emetic). 2.8. Statistical data analysis Reproducibility of the multiplex PCR was tested by determination of intra-assay (results of the same sample in different tubes in the same run) and inter-assay (results of the same sample in different runs) variation of crossing points and melting temperatures for a test panel of 10 B. cereus strains (5 emetic, 5 enterotoxic). The coefficient of variance (CV ¼ SD/arithmetic mean of Ct or Tm) was calculated for results of three replicates in the same run (intraassay) and for results of replicates in three separate runs (interassay). 3. Results
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PCR assay was high. Melt curves obtained for each reference strain were in agreement with the toxigenic profiles of the reference strains as previously reported (Forghani, Kim & Oh, 2014, Forghani, Seo, & Oh, 2014; Kim et al., 2010, 2012), confirming the specificity of the developed assay (Fig. 1A and B). All the B. cereus group members carrying toxin genes as previously reported were also detected (Fig. 1C and D). Finally, the assay successfully detected all the 23 wild B. cereus used in the study which were previously reported as enterotoxigenic strains (Forghani, Kim & Oh, 2014). In addition, when performing the assay on the DNA suspension containing equal amounts of DNA from B. cereus ATCC 12480 enterotoxic strain and F4810/72 emetic reference strain, 5 distinguishable peaks representing all the five toxin genes present in the reaction tube (ces, cytK, entFM, hblD, nheA) were produced (Fig. 2). Also, the presence of non-target L. monocytogenes did not change the performance of the assay (Fig. 3). 3.3. Sensitivity of the assay The developed pentaplex assay was found to be working efficiently over a DNA concentration range of 20 nge200 fg/reaction (Fig. 4). However, cytK and hblD primers showed a 10-fold higher sensitivity up to 20 fg of DNA per reaction. The obtained standard curve for each targeted gene with its respective regression coefficient (R2 value) is represented in Fig. 5. For further validation of the assay sensitivity, DNA isolated from decimally diluted broth cultures of B. cereus ATCC 12480 and B. cereus F4810/72 with a starting cell concentration of 1.6 106 CFU/mL and 3.3 106 CFU/mL, respectively. The results showed limit of detection (LOD) to be 1.6 102 CFU/mL and 3.3 102 CFU/mL for B. cereus ATCC 12480 and B. cereus F4810/72, respectively. All targets were successfully detected at this cell concentration.
3.1. Pentaplex RT- PCR high resolution melt (HRM) curve assay
3.4. Detection of B. cereus in artificially spiked food samples
The suitability of MeltDoctor™ HRM master mix for the multiplex detection of B. cereus emetic and enterotoxic strains was evaluated. Due to the efficiency of primers, use of saturating DNA binding SYTO9 dye in the MeltDoctor™ HRM master mix and application of slowest possible ramp rate (gradual temperature increments of 0.1 ºC/s), the melt curve peaks obtained for each target were equally sharp, regardless of their product size and GC content. The average melting temperatures (Tm) of all amplicons generated from the targeted genes in 5 reference strains are shown in Table 3.
The efficiency of the developed assay was further validated using 6 different food types. Table 4 shows the LOD of the method in food samples with or without enrichment. The developed multiplex assay was able to detect as low as 2.7 103 CFU/g in the food samples without an enrichment step using the DNeasy® mericonTm Food Kit. However, detection using PrepMan® Ultra Sample Preparation Reagent required 10-fold more organisms than in DNeasy for DNA extraction (Table 4). Following enrichment isolated DNA samples were diluted (1:2) using sterile nuclease-free distilled water. The RT- PCR assay could detect all of the target genes in the multiplex reaction with an LOD of 2.7 101 CFU/g with a 7 h enrichment period, regardless of the DNA extraction method used. Components of each food sample tend to affect the efficiency of DNA extraction/RT- PCR. Thus, samples with less complex matrices or lower amounts of fat or protein gave a lower Ct compared to more complex samples with the same level of contamination. The obtained Ct values of each primer in different foods in uniplex format are shown in Table 5. All foods were spiked at a level of 2.7 105 CFU/g B. cereus.
3.2. Applicability and specificity of the assay Assays were conducted to test the applicability and specificity of the multiplex RT- PCR HRM analysis using a concentration of 20 ng of DNA from microbial strains listed in Table 1. While all the target genes were amplified with Ct values in the range of 11e15, all the non-target strains were undetected (Ct values > 35) by the PCR machine, although 20 ng concentration of DNA employed in the
Table 3 Melting temperature (Tm) of amplicons in 8 B. cereus reference strains. Target gene
B. cereus strains
Mean Tm (ºC)
ces cytk nheA entFM hblD
B. B. B. B. B.
72.20 74.23 76.55 78.42 81.90
cereus cereus cereus cereus cereus
F4810/72, JNHE 36, JNHE 80, JNHE 60, JNHE 95 ATCC 12480, KCTC 1013, KCTC 1092, KCTC 1094, KCTC 1526 ATCC 12480, ATCC 13061, KCTC 1092, KCTC 1094, KCTC 1526 ATCC 12480, KCTC 1013, KCTC 1092, KCTC 1094, KCTC 1526 ATCC 12480, KCTC 1013, KCTC 1092, KCTC 1094, KCTC 1526
± ± ± ± ±
0.24 0.52 0.39 0.29 0.41
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Fig. 1. Melt curve peaks for the simultaneous detection of toxin genes in B. cereus reference strains: A) Detection of all 4 enterotoxin genes (cytK, entFM, hblD, nheA) of B. cereus ATCC 12480; B) Detection of all 4 enterotoxin genes (cytK, entFM, hblD, nheA) of B. cereus KCTC 1013; C) Detection of all 2 enterotoxin genes (cytK, entFM) of B. weinheistephanensis KACC 12001; D) Detection of entFM gene in B. pseudomycoides KACC 12098.
3.5. Reproducibility of the assay Results obtained from a panel of 10 B. cereus strains were used to assess intra-assay and inter-assay variation of the developed approach. Three replicates of each strain were applied in one run and three separate runs revealed average CV values for Tm of 0.025% and 0.06% and for Ct of 0.56% and 1.34%, respectively. 4. Discussion In recent years, melt curve-based RT- PCR assays have emerged as powerful tools for the detection of microbial pathogens ndez-No et al., 2011; Nam, Srinivasan, Gillespie, Murinda, & (Ferna Oliver, 2005; Newby, Hadfield, & Roberto, 2003; Price, Smith, Huygens, & Giffard, 2007), including B. cereus (Wehrle, Didier, €rtlbauer, 2010). In this study, we develMoravek, Dietrich, & Ma oped a novel multiplex RT- PCR high resolution melt curve assay for the simultaneous detection of emetic and enterotoxin producing B. cereus in food samples. The developed assay showed a sensitivity of 200 fg/reaction (25e30 genomic equivalents) for pure DNA, 103 CFU/g for spiked food without enrichment and a
sensitivity of approximately 101 CFU/g after a 7 h enrichment period for spiked food samples. The LODs obtained were enough for the analysis of B. cereus contaminated food without enrichment since contamination levels below 103 CFU/g are considered nchez, Garay, & Aznar, safe for consumers (Martínez-Blanch, Sa 2011). However, typical cell counts in food samples associated with food poisoning are usually much higher in the range of 105e108 CFU/g of B. cereus. The LOD obtained in this study was lower than those of some conventional PCR assays previously developed (Ehling-Schulz et al., 2004; Martínez-Blanch et al., 2011). Kim et al. (2012) and Zhang et al. (2014) also reported a LOD of 103 in different food matrices. Despite their advantages, all of the above mentioned methods need post-PCR procedures such as staining, gel electrophoresis and visualization which are not only time consuming and expensive but also may cause the risk for post-PCR contamination of the samples reducing the efficiency and simplicity of these conventional methods. On the other hand, RT- PCR allows high-throughput results with easy automation and does not require post-PCR detection procedures while it gives similar or higher sensitivity (Wehrle et al., 2010). Food samples artificially contaminated with B. cereus
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Fig. 2. Melt curve peaks for the simultaneous detection of all 4 enterotoxin genes (cytK, entFM, hblD, nheA) and emetic toxin gene (ces) in emetic/enterotoxic DNA suspension (equal amounts of DNA from B. cereus ATCC 12480 enterotoxic and F4810/72 emetic reference strain) by the pentaplex RT- PCR high resolution melt (HRM) curve assay.
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Fig. 4. Sensitivity of the multiplex assay in detecting B. cereus ATCC 12480 at DNA concentrations ranging from 20 ng to 200 fg using MeltDoctor™ HRM master mix.
concentrations lower than 3 log CFU/mL were not detected without enrichment, thus necessitating the addition of an enrichment step for lower concentrations. A LOD of approximately 101 CFU/g was achieved after an enrichment period of 7 h, which is similar to some
Fig. 3. Melt curve peaks of all 4 enterotoxin genes (cytK, entFM, hblD, nheA) of B. cereus ATCC 12480 detection using 20 ng of the target DNA with and without 20 ng of L. monocytogenes non-target DNA as background (A); Detection of all 4 enterotoxin genes of B. cereus ATCC 12480 using 200 fg of the target DNA with and without 20 ng of L. monocytogenes non-target DNA as background (B).
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Ct
28 26
ces
24
cytK
22
entFM
20 18
hblD
16
nheA
14 12 10 20 ng
2 ng
200 pg
20 pg
2 pg
200 fg
20 fg
DNA concentraƟon/reacƟon Fig. 5. Standard curve for all 5 target genes: ces (R2 ¼ 0.9937), cytK (R2 ¼ 0.9877), entFM (R2 ¼ 0.9772), hblD (R2 ¼ 0.9911) and nheA (R2 ¼ 0.9739) constructed using decimally diluted DNA.
previous reports (Forghani, Seo & Oh, 2014) and much shorter than the works of Wehrle et al. (2010) and Alarcon, Ohta, and Yokoyama (2005). Priha et al. (2004) reported a RT- PCR assay for the detection of B. cereus using 16SrDNA gene. Fricker et al. (2007) developed a RT-
PCR assay for the detection of emetic B. cereus using ces primer. nez-Blanch et al. (2009) developed a real-time PCR Whereas, Martıa assay for the detection and quantification of enterotoxigenic B. cereus which could not detect the emetic strains and its toxin nez-Blanch et al. (2010) detection range was limited. Later, Martıa developed a RT- PCR assay for detection and quantification of B. cereus group spores in food. Wehrle et al. (2010) developed a melt curve based RT- PCR approach for the detection of enteropathogenic B. cereus. However, the developed approach did not include entFM gene which is the most frequent toxin gene along with nhe in the majority of toxigenic B. cereus strains (Ngamwongsatit et al., 2008; Tewari, Singh, & Rashmi, 2013). More recently, Fern andeznez-Blanch et al. (2011) developed No et al. (2011) and Martıa real-time assays for the detection of B. cereus and viable B. cereus, respectively, without the ability to discriminate any of toxin genes. Finally, Dzieciol et al. (2013) developed a quantitative real-time approach for detection and differentiation of emetic (using ces gene) and non-emetic B. cereus. Each of the above mentioned approaches has its advantages but some of them do not detect any of the toxin genes responsible for causing B. cereus food poisoning. This is important due to the possible absence of toxin genes in some B. cereus strains has been nez et al., 2013; previously reported (Forghani, Seo & Oh, 2014; Jime Priha et al., 2004). Therefore, any detection approach without the ability of detecting toxin genes may detect non-pathogenic strains which are of no harm to the consumers. On the other hand, the
Table 4 Multiplex real-time PCR results of artificially contaminated food samples with/without enrichment using two different DNA extraction methods. Food sample
Spiked B. cereus concentration (CFU/g)
Real-time PCR result after enrichment DNeasy® mericonTm food kit
Infant formula
Milk
Kimbab
Rice
Pasta
Tteok
2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7
101 102 103 104 105 106 101 102 103 104 105 106 101 102 103 104 105 106 101 102 103 104 105 106 101 102 103 104 105 106 101 102 103 104 105 106
PrepMan® ultra sample preparation reagent
0h
3h
5h
7h
0h
3h
5h
7h
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
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Table 5 Mean Ct values of each PCR primer in uniplex format, using different foods spiked with 2.7 105 CFU/g B. cereus. Target gene
Mean Ct value Rice
ces cytK entFM hblD nheA
17.52 15.74 19.21 18.38 18.64
Tteok ± ± ± ± ±
0.11 0.09 0.07 0.08 0.18
18.13 16.11 19.27 18.55 18.97
Pasta ± ± ± ± ±
0.09 0.09 0.27 0.22 0.17
18.32 16.29 19.46 18.64 19.34
± ± ± ± ±
0.26 0.17 0.24 0.28 0.16
Infant formula
Milk
± ± ± ± ±
19.15 16.81 19.95 19.52 20.29
18.76 16.58 19.63 18.97 19.69
0.83 0.46 0.39 0.31 0.38
Kimbab ± ± ± ± ±
0.76 0.22 0.14 0.33 0.25
19.47 17.93 20.88 20.47 21.16
± ± ± ± ±
0.38 0.61 0.51 0.39 0.22
Values are means of 3 measurements ± SD.
other reported methods lacked the ability to detect one or more of the major enterotoxins (cytK, entFM, hblD, nheA) or emetic toxin (ces). Developing detection methods for various types of toxins from enteropathogenic B. cereus in food samples are very important as there is a high variation in the distribution of toxin genes among B. cereus strains and several toxigenic profiles are available (Forghani, Kim & Oh, 2014). Also, considering the gene polymorphism which may occur in the toxin genes of wild B. cereus strains, a higher number of multiplexing experimental designs will strongly increase the detection rate in case of probable primer mismatch for some of the target genes (Schoeni & Wong, 1999; Thaenthanee, Wong, & Panbangred, 2005). Therefore, the present approach was developed to simultaneously detect all the four major enterotoxin genes as well as the emetic toxin genes in a single reaction. Food samples contain many organic and inorganic compounds, such as enzymes, polysaccharides, proteins and salts (Forghani, Seo , & Skapova, & Oh, 2014; Spanov a, Rittich, Karpískov a, Cechov a 2000). However, none of these inhibitors can be detected using the routine spectrophotometric measurements of DNA samples. This explains the reduction in LOD along with complexity of the food matrix as well as shorter enrichment time for foods with simpler matrix. Thus, application of an appropriate DNA extraction method capable of maximum recovery and inhibitor removal is a critical point for achieving high sensitivity of the method. As expected, this study showed a ten times higher sensitivity for the realtime approach using a specific (DNeasy® mericonTm) DNA extraction kit compared to PrepMan® Ultra Sample Preparation Reagent (Table 4). However, the latter has the advantage of shorter DNA extraction time less than 20 min compared to approximately 2 h needed for DNeasy® kit, making it the best choice for DNA isolation from bacterial cultures or food samples with higher bacterial loads (e.g. longer enrichment times). Other measures that could reduce the level of PCR inhibitors include a low speed centrifugation step to sediment large solid compound in the food homogenate upper liquid part (Singh & Mustapha, 2014) which we added to the routine DNA extraction protocols for both kits in this study. Probe-based methods are usually more specific than melt curve real-time PCR assays, but the specificity of the melt curve assays can also be maximized via appropriate primer design. Furthermore, melt curve based assays can be more sensitive (Fricker et al., 2007) and have a much lower running cost (Singh & Mustapha, 2014). As the probe itself has a limited storage life compared to the primers and master mix, it is also difficult to optimize for high multiplex numbers (such as our pentaplex approach) due to real-time chemistry as well as the necessity of multiple detection channels in the real-time PCR instrument. Thus, intercalating dyes such as SYTO9 have become the preferred choice and are being increasingly used for bacterial identification (Eischeid, 2011; Tong & Giffard, 2012). In this study, SYTO9 was selected over SYBR® Green I due to its several advantages. SYBR® Green I has a number of limitations that include the inhibition of PCR, preferential binding to GC-rich sequences and more noise production during the melt curve
analysis (Dragan et al., 2012; Giglio, Monis, & Saint, 2003; Karsai, Müller, Platz & Hauser, 2002; Nath, Sarosy, Hahn, & Di Como, 2000; Reed, Kent, & Wittwer, 2007). On the other hand, SYTO9 being a saturating DNA binding dye does not inhibit PCR and also does not show preferential PCR amplicon binding making it a better choice for multiplexing method (Monis, Giglio, & Saint, 2005). Most importantly, it provides the possibility of high resolution melt curve analysis which was of great importance in this work because of the close Tm of 5 different amplicons present in the same reaction tube (amplicon separated by approximately 2 C difference from each other) and the ability of SYTO9 to distinguish between amplicons with close Tm. However, in order to maximize the assay resolution, the default 0.3 C/s ramp rate (melting step) of StepOne™ real-time PCR machine was manually changed to 0.1 C/s resulting in much slower amplicon melting and generation of data with much higher resolution. In conclusion, although traditional culture based methods are still the gold standard for the detection of B. cereus, they require about 3 days (Martínez-Blanch et al., 2009) in food for confirming the presence of B. cereus. The current assay revealed a detection limit of 103 CFU/g or higher in less than 6 h, but with an enrichment step of 7 h it could detect contamination levels as low as 101 CFU/g food and does not require time-consuming and contaminationprone post-PCR steps. It provides additional information on the toxigenic profiles of the detected strains. This advantage makes the present approach a candidate biomarker for other applications such as in clinical settings. The pentaplex RT- PCR high resolution melt curve assay described in this study offers a simple workflow with a total run time of less than 12 h, with such a wide detection range of both emetic and enterotoxic strains of B. cereus. It is an economical alternative to the previous methods both for the melt curve analysis technology instead of the probe-based technology for real-time detection and the high number of multiplexing covering 5 different toxin genes in a single reaction. Acknowledgments This research was supported by Bio-industry Technology Development Program (112137-3) Ministry of Agriculture, Food and Rural Affairs. References Abdou, M. A., Awny, N. M., & Abozeid, A. A. E. M. (2011). Prevalence of toxicogenic bacteria in some foods and detection of Bacillus cereus and Staphylococcus aureus enterotoxin genes using multiplex PCR. Annals of Microbiology, 62(2), 569e580. Agata, N., Mori, M., Ohta, M., Suwan, S., Ohtani, I., & Isobe, M. (1994). A novel dodecadepsipeptide, cereulide, isolated from Bacillus cereus vacuole formation in Hep-2 cells. FEMS Microbiology Letters, 121, 31e34. Alarcon, B., Ohta, M., & Yokoyama, K. (2005). PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food. Journal of Applied Microbiology, 100, 352e364. Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N., & Patel, H. R. (2005). Basic principles of real-time quantitative PCR. Expert Review of Molecular Diagnostics, 5, 209e219.
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