Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

A simple, rapid, cost-effective and sensitive method for detection of Salmonella in environmental and pecan samples S. Dobhal1, G. Zhang2, C. Rohla3, M.W. Smith4 and L.M. Ma1 1 National Institute for Microbial Forensics and Food and Agricultural Biosecurity, Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, USA 2 Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA 3 Samuel Robert Noble Foundation, Ardmore, OK, USA 4 Department of Horticulture and Landscape Architecture, Oklahoma State University, Stillwater, OK, USA

Keywords detection, feces, inhibitors, PCR, pecan, Salmonella, soil. Correspondence Li Maria Ma, National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA. E-mail: [email protected] 2014/0537: received 16 March 2014, revised 6 June 2014 and accepted 16 June 2014 doi:10.1111/jam.12583

Abstract Aims: PCR is widely used in the routine detection of foodborne human pathogens; however, challenges remain in overcoming PCR inhibitors present in some sample matrices. The objective of this study was to develop a simple, sensitive, cost-effective and rapid method for processing large numbers of environmental and pecan samples for Salmonella detection. This study was also aimed at validation of a new protocol for the detection of Salmonella from inshell pecans. Methods and Results: Different DNA template preparation methods, including direct boiling, prespin, multiple washing and commercial DNA extraction kits, were evaluated with pure cultures of Salmonella Typhimurium and with enriched soil, cattle feces and in-shell pecan each spiked individually with Salmonella Typhimurium. PCR detection of Salmonella was conducted using invA and 16S rRNA gene (internal amplification control) specific primers. The effect of amplification facilitators, including bovine serum albumin (BSA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) and gelatin on PCR sensitivity, was also evaluated. Conducting a prespin of sample matrices in combination with the addition of 04% (w/v) BSA and 1% (w/v) PVP in PCR mix was the simplest, most rapid, cost-effective and sensitive method for PCR detection of Salmonella, with up to 40 CFU Salmonella per reaction detectable in the presence of over 109 CFU ml1 of background micro-organisms from enriched feces soil or pecan samples. Conclusions: The developed method is rapid, cost-effective and sensitive for detection of Salmonella from different matrices. Significance and Impact of the study: This study provides a method with broad applicability for PCR detection of Salmonella in complex sample matrices. This method has a potential for its application in different research arenas and diagnostic laboratories.

Introduction Foodborne illnesses caused by pathogenic bacteria constitute a major public health concern with estimates of 48 million illnesses, 128 000 hospitalizations and 3000

deaths occurring annually in the United States (CDC 2011a). Salmonella is one of the human pathogens associated with a large number of foodborne illness outbreaks in the United States (CDC 2013), including several large multistate outbreaks in the past 10 years. Recent

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multistate outbreaks of foodborne illnesses associated with consumption of tree nuts (CDC 2009, 2011b) have increased the attention on food safety issues related to tree nut production. Past surveys and recalls have also indicated the association of Salmonella with tree nuts (Little et al. 2010; Palumbo et al. 2011; Lambertini et al. 2012). Tree nuts are dry commodities and may not support the growth of foodborne human pathogens; however, these pathogens can survive and cause illness when present on nuts, even in low numbers (Podolak et al. 2010). The sources of nut contamination are not clearly understood, but likely come from the preharvest environment or harvest/postharvest handling (Danyluk et al. 2008; Lambertini et al. 2012). One step in tree nut (such as pecan) harvesting is to shake the nuts to the ground and then collect them by machine; these nuts could come into contact with pathogenic bacteria present in the soil or in animal feces on the floor of the grove (Patton et al. 2009). More information is needed about the natural occurrence and potential sources or contributing factors leading to tree nut contamination by human pathogens. Therefore, a reliable tool for the detection of these pathogens in environmental samples is critical for monitoring the contamination in such environments. Since the 1980s, polymerase chain reaction (PCR) has become a valuable method for the detection of microorganisms in many food and environmental sample matrices because of its speed, sensitivity, specificity and low cost when compared to conventional culture methods (Wang et al. 2007). However, PCR detection of pathogens in environmental samples, such as soil, fecal material and/or complex commodities, is still challenging, often requiring sophisticated extraction procedures and further purification steps to remove substances/compounds that are inhibitory to PCR (Josephson et al. 1991; Stacy-Phipps et al. 1995). These DNA preparation steps are often tedious, time-consuming and expensive. The aim of this study was to develop a simple, rapid, costeffective and sensitive PCR method for the detection of Salmonella in environmental samples. We chose to target the invA gene, which is located on pathogenicity island 1 of Salmonella spp. and is required for bacterial invasion into epithelial cells (Malorny et al. 2003; Mercanoglu and Griffiths 2005). This gene is highly conserved in almost all known serovars of Salmonella (Rahn et al. 1992; Chiu and Ou 1996; Swamy et al. 1996) and has been used widely for the detection of Salmonella. In this study, we evaluated eight different DNA template preparation methods in their speed, cost and sensitivity for the PCR detection of Salmonella. A primer pair specific to 16S rRNA gene was included as an internal amplification control (IAC). To our knowledge, this is the first vali1182

dated protocol for the preparation of DNA templates from in-shell pecans for the PCR detection of a human pathogen. Materials and methods Bacterial strain, media and culture conditions Salmonella enterica subspecies enterica serovar Typhimurium, a cantaloupe isolate available in our strain collection, was maintained on trypticase soy agar (TSA; BBL, Becton Dickinson, Sparks, MD) at 4°C. Cultures were activated by transferring a loopful of inoculum into 5 ml of trypticase soy broth (TSB; BBL) and incubating the culture at 37°C overnight with shaking at 150 rev min1. Serial dilutions of the overnight cultures were prepared in sterile 01% (w/v) peptone water (BBL, Becton Dickinson, Sparks, MD) to obtain the desired titres, which were confirmed by plating on TSA and incubation at 37°C for 24 h. The cell counts were recorded and calculated in terms of log10 CFU ml1. Environmental samples and their overnight enrichment The soil and cattle fecal samples were obtained from the Oklahoma State University Research Station in Central Oklahoma, and native in-shell pecans [Carya illinoinensis (Wangenh.) K. Koch] were obtained from a Research Station in Southern Oklahoma. Soil or cattle feces (10 g) were suspended separately in 90 ml of TSB contained in a sterile filtered whirl-pak bags (24 oz) (Thermo Fischer Scientific Inc., Waltham, MA) (He et al. 2011). Similarly, approx. 50 g of in-shell pecans (7–9 pecans) was added to 200 ml of TSB, in sterile filter-containing whirl-pak bags (Danyluk et al. 2007). Each bag containing either the soil or fecal suspension was homogenized in a stomacher (Seward Model 400C, London, UK) at 260 rev min1 for 2 min. Each bag containing in-shell pecans was shaken by hand for 1 min, the surface of pecans was then massaged by hand for 2 min and each bag was again shaken by hand for 1 min. The pecans were allowed to soak in the above suspension for 30 min at room temperature; the rinse fluid was withdrawn from the bag and transferred to a sterile 500 ml bottle. Each of the suspension (soil/feces/pecan) was incubated at 42°C for 24 h. To obtain a uniform suspension for DNA template preparation, the soil/fecal enrichments were homogenized in stomacher for 1 min at 260 rev min1; the bottles containing enriched wash fluid from pecan surface were shaken for 1 min at 150 rev min1, and then, 1 ml of the enrichment suspension was withdrawn from each of the samples. These enrichments were also used in spiking experiments as described below.

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DNA template preparation methods Overnight pure cultures of Salmonella, or soil or fecal, or pecan enrichments spiked with Salmonella (final titre: 108 CFU ml1) were subjected to the following DNA template preparation methods: (i) Direct method-the culture suspensions were heated at 100°C for 12 min. This crude cell lysate was centrifuged at 15 000 g for 3 min at 4°C, and the supernatant served as DNA template for PCR; (ii) prespin method-the culture suspensions were centrifuged at 500 g, for 1 min at 4°C, the supernatant was taken, centrifuged at 15 000 g, and the pellet was suspended in 1 ml of autoclaved distilled water. This suspension was heated at 100°C for 12 min in dry bath followed by centrifugation at 15 000 g for 3 min at 4°C, and the supernatant served as template for PCR; (iii) direct centrifugation-the culture suspensions were directly centrifuged at 15 000 g for 3 min at 4°C, the pellet was re-suspended in 1 ml of distilled water, followed by heat treatment at 100°C for 12 min in dry bath, centrifuged at 15 000 g for 3 min at 4°C, and the supernatant was used as template for PCR; (iv) direct centrifugation with a wash-the culture suspensions were centrifuged at 15 000 g for 3 min at 4°C, the pellet was washed with 19 phosphate-buffered saline (PBS), followed by centrifugation at 15 000 g for 3 min, re-suspension of the pellet in 1 ml autoclaved water and heat treatment as described in direct centrifugation method; (v) InstaGene Matrix method-the culture suspensions were centrifuged at 15 000 g for 1 min, and 200 ll of InstaGene Matrix (Bio-Rad, Hercules, CA) was added to the pellet, followed by incubation at 56°C for 30 min. The suspension was vortexed at high speed for 10 s, followed by placing the tubes at 100°C for 8 min in dry bath and vortexing the suspension again at high speed for 10 s. The suspension was finally centrifuged at 15 000 g for 3 min. The supernatant was used as a template in PCR; (vi) DNeasy Blood and Tissue DNA Extraction Kit (QIAGEN, Valencia, CA) method-the manufacturer’s instruction was followed which included cell lysis at 56°C for 30 min, purification and elution of DNA by mini spin column, in addition to other steps; (vii) PowerFood Microbial DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA)-this method was only used to prepare DNA templates from spiked pecan enrichment suspensions. The manufacturer’s instruction was followed which consisted of multiple steps including the use of microbead tubes and spin filters; (viii) PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc.)-this method was only used to prepare DNA templates from spiked soil enrichment suspensions. The protocol was similar to PowerFood Microbial DNA Isolation Kit. Two microliter of template DNA was used from all

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preparations except for InstaGene preparation of which 4 ll was used for PCR. PCR primers and amplification conditions Previously validated (Rahn et al. 1992) PCR primers targeting the invA gene of Salmonella consisted of the forward primer, 139 (50 -GTGAAATTATCGCCACGTTCGGG CAA-30 ), and reverse primer, 141 (50 -TCATCGCACCGTC AAAGGAACC-30 ), amplifying a 284-bp fragment. Another set of primers targeting the 16S rRNA gene was also used as internal amplification control in the reaction, with forward primer VMP5 (50 -AGAAGCACCGGCTA ACTC-30 ) and reverse primer VMP6 (50 -CGCATTT CACCGCTACAC-30 ), amplifying a 204-bp fragment (Monday et al. 2007). All primers were synthesized by Integrated DNA Technologies (Coralville, IA) and were validated for their specificity in silico against available sequences of Salmonella in the Genbank database using BLASTn (Altschul et al. 1990). The final multiplex PCR mix volume was 20 ll, containing 2 ll of DNA template, 5 lmol l1 of each forward and reverse invA primers and 25 lmol l1 of each forward and reverse 16S primers, 10 ll of GoTaq Green Master Mix (29 GoTaq Green reaction buffer, 400 mmol l1 of each of four deoxynucleoside triphosphates and 3 mmol l1 MgCl2; Promega, Madison, WI) and 15 mmol l1 MgCl2. PCR was performed in an Eppendorf thermal cycler (Eppendorf, Hauppauge, NY) with cycling conditions consisted of initial denaturation at 94°C for 3 min and 35 cycles of the following: denaturation at 94°C for 30 s, annealing at 60°C for 45 s and extension at 72°C for 30 s, with a final extension at 72°C for 7 min. PCR amplicons were visualized on 15% agarose gels containing SYBR Safe DNA gel stain (Invitrogen, Carlsbad, CA). A positive control (pure genomic DNA from Salmonella Typhimurium) and a negative template control (nuclease-free water) were included in each PCR assay. Addition of amplification facilitators to the PCR mixture To counteract the effect of potential PCR inhibitors in environmental sample DNA template preparations, combinations of different amplification facilitators (AFs) were added to the PCR mixtures. Combinations of AFs included 01% BSA (w/v) + 1% PVP (w/v), 04% BSA (w/v) + 1% PVP (w/v), 01% gelatin (w/v) and 04% PEG (w/v) (Xin et al. 2003; Rudi et al. 2004). For comparison among the different DNA template preparation protocols, PCR assays were performed with and without the AFs, using templates prepared from artificially inoculated (108 CFU ml1 of Salmonella) enriched sample matrices.

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Sensitivity of PCR on spiked environmental samples To test whether the presence of other niche-sharing micro-organisms would affect PCR sensitivity, an overnight pure culture of Salmonella (approx. 109 CFU ml1) was serially diluted 10-fold with each of the enrichment suspension from pecan or soil or feces and DNA templates were prepared from dilutions of 107–101 CFU ml1 following the prespin method described above. The cell populations in the pure culture and enrichment suspensions were determined by directly plating. The effectiveness of the AFs in PCR was also evaluated using the prespin method of template preparation. Results Sensitivity of PCR assays The detection limit of the multiplex PCR using 10-fold serially diluted crude cell lysate of an overnight pure culture of Salmonella Typhimurium prepared by method 1 (direct) or 2 (prespin) was approx. 104 CFU ml1 or 40 CFU per reaction (Fig. 1). No amplification was observed with DNA templates prepared from overnight enrichment suspensions of feces or soil that were artificially spiked with 108 CFU ml1 of Salmonella (data not shown). However, amplicons of expected size (284 bp for the invA gene and 204 bp for the 16S rRNA gene) were observed on DNA templates prepared from overnight enrichment suspensions of pecans that were artificially spiked with 108 CFU ml1 of Salmonella, with a sensitivity of 104 CFU ml1 or 40 CFU Salmonella/reaction (data not shown). Comparison of DNA preparation methods using spiked environmental (fecal or soil) and pecan samples Pure genomic DNA of Salmonella was extracted from an overnight pure culture of Salmonella using the DNeasy

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Figure 1 Multiplex PCR Sensitivity assay using 10-fold serial dilution of crude cell lysate of Salmonella. Lane M, 100-bp ladder; lane 1–8, Decreasing cellular concentrations of 108–101 CFU ml1 of crude cell lysate of Salmonella; lane 9—NTC, nontemplate control.

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Blood and Tissue Kit and used as a positive control in all assays performed. For enriched feces that were artificially spiked with 108 CFU ml1 of Salmonella, a faint band corresponding to IAC was observed when using the DNA template prepared by method 2 (prespin method, Fig. 2a, lane 4) and method 6 (Blood and Tissue Kit, Fig. 2a, lane 8), suggesting that these two methods might be useful for the preparation of DNA templates. No amplification was observed for artificially inoculated enriched fecal samples (EFS) following DNA extraction by the other methods tested. For enriched pecan samples (EPS), expected PCR amplification was observed from all seven methods tested (Fig. 2b), indicating that all of these methods are suitable for template preparation. In contrast, for inoculated enriched soil samples (ESS), PCR amplification was observed only from DNA prepared with Power Soil Mo Bio Kit (Fig. 2c, lane 8). No amplification was observed using DNA from other methods tested. The experiment was performed three times and produced similar results. The effect of amplification facilitators among DNA preparation methods The combinations of PCR facilitators including BSA and PVP, gelatin and PEG alone were added to PCR mixtures to evaluate their effect on amplification, using template DNA prepared from different sample matrices prepared using different methods (Figs 3 and 4). For fecal samples, the addition of 01% BSA + 1% PVP to PCR mixture improved PCR performance, resulting in amplicons of expected size with templates prepared using all methods except method 1, where no amplification corresponding to the invA gene was observed (Fig. 3a left panel, lane 2). However, the use of 04% BSA + 1% PVP in PCR resulted in bands of higher fluorescence intensity when compared with the other AF combinations tested (Fig. 3a right panel). When 01% gelatin was added to the PCR mix, no amplification of target genes was observed with any of the methods tested except for method 2 with less fluorescence intensity compared to the first two AFs (Fig. 3b left panel, lane 3) and method 6 in which only a band corresponding to 16S gene amplicon was observed (Fig. 3b left panel, lane 7). When testing, 04% PEG in the PCR mix bands of expected size corresponding to the invA and 16S gene was only observed with DNA prepared by method 6 (Fig. 3b right panel, lane 7). In addition, PCR performed with DNA prepared using the other methods either failed or produced a single band of 204 bp, corresponding to 16S gene (Fig. 3b right panel). Among the AFs tested, the combination of 04% BSA + 1% PVP gave the best performance/improvement in PCR with all six extraction methods tested. Therefore,

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Figure 2 Multiplex PCR performed using DNA templates prepared by different DNA extraction methods from different sample matrices spiked with overnight grown culture of Salmonella (109 CFU ml1). (a) Amplification of DNA from spiked fecal enrichment culture: lane M, 100-bp ladder; lane 1, positive control; lane 2, DNA extracted from overnight grown culture of Salmonella by method 6 (positive control); lanes 3, 4, 5, 6, 7 and 8, DNA extracted by methods 1, 2, 3, 4, 5 and 6, respectively; lane 9, nontemplate control (water). (b) Amplification of DNA from spiked pecan enrichment culture: lane M, 100-bp ladder; lane 1, positive control; lanes 2, 3, 4, 5, 6, 7 and 8, DNA extracted by methods 1, 2, 3, 4, 5, 6 and 7, respectively; lane 9, nontemplate control (water). (c) Amplification of DNA from spiked soil enrichment culture: lane M, 100-bp ladder; lane 1, positive control; lanes 2, 3, 4, 5, 6, 7 and 8, DNA extracted by methods 1, 2, 3, 4, 5, 6 and 8, respectively; lane 9, nontemplate control (water).

this combination of AFs was included in subsequent PCR mixes using DNA prepared from spiked fecal samples. Amplicons of expected size were observed with all seven methods of DNA preparation from spiked pecanenriched samples, indicating the absence of inhibitors in pecan enrichment cultures, and the inclusion of 04% BSA + 1% PVP in these PCR mixes provided no benefit except for an increase in the fluorescence intensity (data not shown). Similarly, addition of these AFs was also tested with soil enrichment cultures spiked with Salmonella and DNA templates prepared using the different extraction methods. When using 01% BSA + 1% PVP, an expected amplification was observed with DNA

templates prepared by all the methods except for methods 1 and 4 (Fig. 4a left panel) with no amplification corresponding to invA gene (Fig. 4a left panel, lanes 2 and 5). However, use of 04% BSA + 1% PVP led to improvement in PCR amplification with all seven methods, producing higher fluorescence intensity compared with the 01% BSA + 1% PVP treatment (Fig. 4a right panel). Similarly, the addition of gelatin or 04% PEG provided no protection from inhibitors, and no amplification was observed when using DNA templates prepared using all of the extraction methods except for method 7 (the Power Soil Mo Bio Kit) (Fig. 4b left and right panels, lane 8). We selected the prespin method (method 2) of template preparation, as it is simple to perform and produce similar or better results than the other methods tested. Sensitivity of PCR assays using templates prepared using the prespin method in combination with AFs (04% BSA + 1% PVP) Further PCR sensitivity assays were performed in different sample matrices using the DNA templates prepared by prespin method in combination with and without 04% BSA + 1% PVP in the reaction. No amplification was observed when sensitivity assays were performed without the AFs (04% BSA + 1% PVP), with DNA templates prepared from spiked EFSs and ESSs (Fig. 5a,b, both left panels). In contrast, expected amplifications were observed, and detection limits reached 104 CFU ml1 or 40 CFU per reaction in each of sample matrices (EFS and ESS) after the addition of 04% BSA + 1% PVP to the reaction mixture (Fig. 5a,b, both right panels). This detection limit was similar to that in the assays performed with crude cell lysate of overnight pure cultures of Salmonella. Therefore, the combination of template preparation using method 2 with inclusion of 04% BSA + 1% PVP in PCR mixture was selected for detecting the pathogen from the fecal and soil matrices. There was no effect of AFs on the detection limit of PCR in spiked EPS. The detection limit of sensitivity assays performed with spiked EPS was also 104 CFU ml1 or 40 CFU per reaction (Fig. 5c). Discussion Although PCR is widely used for the detection of pathogens, it is very challenging to detect pathogens from environmental samples due to the presence of PCR inhibitors. Consequently, complicated steps of purification or expensive commercial DNA extraction kits have been used to prepare DNA templates for PCR from such matrices (Kreader 1996), often resulting in significant loss

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Figure 3 Effect of amplification facilitators on the multiplex PCR performed with DNA extracted by six different DNA extraction protocols from spiked fecal enrichment culture. (a) Inclusion of 01% BSA and 1% PVP (left panel of the gel) and inclusion of 04% BSA and 1% PVP (right panel of the gel). (b) Inclusion of 01% gelatin (left panel of the gel) and inclusion of 04% PEG (right panel of the gel), in the PCR. Lane M, 100-bp ladder; lane 1, positive control; PCR amplification of, lane 2, DNA extracted by method 1; lane 3, DNA extracted by method 2; lane 4, DNA extracted by method 3; lane 5, DNA extracted by method 4; lane 6, DNA extracted by method 5; lane 7, DNA extracted by method 6; lane 8, nontemplate control (water).

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of target DNA (Miller et al. 1999). In this study, we developed a simple, rapid, cost-effective and sensitive method for PCR detection of Salmonella in soil, fecal and pecan samples using a simple DNA template preparation method and PCR amplification facilitators (AFs). This method was suitable for use with artificially inoculated fecal or soil or pecan samples, with which it was demonstrated to be sensitive with detection limit of 40 CFU per reaction for Salmonella cells. Primer pair 139-141, targeting the Salmonella invA gene, was selected for the study because it has been 1186

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Figure 4 Effect of different amplification facilitators on multiplex PCR performed with DNA extracted by seven different DNA extraction protocol from spiked soil enrichment culture. (a) Inclusion of 01% BSA and 1% PVP (left panel of the gel) and inclusion of 04% BSA and 1% PVP (right panel of the gel). (b) Inclusion of 01% gelatin (left panel of the gel) and inclusion of 04% PEG (right panel of the gel), in the PCR. Lane M, 100-bp ladder; lane 1, positive control; PCR amplification of, lane 2, DNA extracted by method 1; lane 3, DNA extracted by method 2; lane 4, DNA extracted by method 3; lane 5, DNA extracted by method 4; lane 6, DNA extracted by method 5; lane 7, DNA extracted by method 6; lane 8, DNA extracted by method 8; lane 9, nontemplate control (water).

widely used in PCR detection of Salmonella in a variety of sample matrices and had been validated by different international laboratories (Rahn et al. 1992; Malorny et al. 2003). The use of IAC is becoming more common in diagnostic PCR assays as it indicates the presence of inhibitors to DNA polymerase and any technical errors that may occur during PCR (Malorny et al. 2004), hence reducing false-negative results (Hoofnar et al. 2003; Malorny et al. 2004). A primer pair targeting the 16S rRNA gene region was selected from the published literature (Monday et al. 2007) and used as IAC in this study; this

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(b) Figure 5 Multiplex sensitivity assay performed using 10-fold serial dilution of Salmonella spiked in different sample matrices processed by method 2 of DNA extraction protocol, and PCR was performed with the template, without (left panel of the gel) and with the inclusion of 04% BSA and 1% PVP (right panel of the gel). (a) Sensitivity assay performed in the fecal enrichment culture. (b) Sensitivity assay performed in the soil enrichment culture. (c) Sensitivity assay performed in the pecan enrichment culture. Lane M, 100-bp ladder; lane 1, positive control; 10-fold decreasing cellular concentration of Salmonella: lane 2, 107 CFU ml1; lane 3, 106 CFU ml1; lane 4, 105 CFU ml1; lane 5, 104 CFU ml1; lane 6, 103 CFU ml1; lane 7, 102 CFU ml1; lane 8, 101 CFU ml1; lane 9, NTC (water).

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primer has a Tm range similar to that of invA primers and produces a distinct amplicon. In our initial tests of PCR sensitivity using 10-fold serial dilutions of crude cell lysate prepared from overnight pure cultures of Salmonella with selected primer pairs, the detection limit was 40 CFU per reaction, which is comparable to the findings reported in previous studies (Rahn et al. 1992; Malorny et al. 2003). Therefore, multiplexing of these two primer pairs did not affect the sensitivity of 139-141 primer set for the detection of Salmonella. Impairment of direct DNA amplification by PCR from an environmental sample occurs due to naturally occurring PCR inhibitors such as bile salts and complex polysaccharides in feces (Lantz et al. 1997; Monteiro et al. 1997), humic substances in soil (Kozdr oj and van Elsas 2000) and tannins on the surfaces of nuts (pecan) (Krause et al. 2001) that could be coextracted with the DNA template (Akane et al. 1994; Eilert and Foran 2009). PCR inhibitors may directly inactivate Taq polymerase by binding to its active site (Opel et al. 2009; Funes-Huacca et al. 2011), thus competing with cofactors responsible for the activity of enzyme, or may bind with other reaction components such as dNTPs or DNA template,

thereby inhibiting PCR amplification. There are several published DNA extraction/preparation and purification methods for removing inhibitors from environmental samples, but these methods are time-consuming or expensive, limiting their use when handling a large number of samples (Stauffer et al. 2008). Furthermore, different DNA extraction methods are required to prepare templates from diverse sample matrices. In this study, a single extraction method was developed for preparing DNA templates from dissimilar sample matrices. The soil-, fecal- or pecan-enriched samples were artificially inoculated with overnight culture of Salmonella, and template DNA was prepared following each method described earlier. We did not observe any amplification using templates prepared from artificially inoculated EFSs, except for the presence of a band corresponding to IAC (16S rDNA) in methods 2 and 6 (Prespin and Blood and Tissue Kit), indicating the presence of inhibitors. Use of these two methods reduced the carry-over of inhibitors compared to the other methods. In contrast, DNA prepared from ESSs could not be amplified, except when prepared by method 8 (soil extraction kit), indicating that the other methods were hampered by PCR

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inhibition. It appeared that the in-shell pecan samples contained lower levels of PCR inhibitors than the fecal and soil samples, as the pathogen was detected with the DNA templates prepared by all extractions except method 4. It has been well documented that incorporation of AFs into PCR mixtures reduces inhibition (Al-Soud and R adstrom 2000; Jiang et al. 2005; Fatima et al. 2011). Therefore, we tested different combination of AFS, including BSA, PVP, gelatin and PEG in PCR mix, for amplification of DNA templates prepared using different protocols from each sample matrix. Among the four AFs tested, only the combination of 04% BSA + 1% PVP resulted in enhanced fluorescence signal in the presence of background microflora. The plausible reason for relieving of PCR from the effect of inhibitors (phenolic compounds, lipids, etc.) is due to the scavenging property of BSA and PVP. Previous study by Kreader (1996) has shown that BSA binds to the phenolic compounds and the lipids which are known to bind and inactivate Taq DNA polymerase. Additionally, BSA acts as an alternative substrate for proteinases (present in sample matrices) thereby protecting Taq DNA polymerase from inactivation. Similarly, PVP also binds to the phenolic groups forming PVP-phenolic complexes, thus removing molecules such as humic acid which contains phenolic groups (Guy et al. 2003). Farell and Alexandre (2012) have reported that BSA is sensitive to high temperature of PCR and may rapidly lose its scavenging abilities. As PVP can withstand high temperature (autoclavable), we speculate the combination of BSA and PVP may reduce the effect of inhibitors on PCR. This finding is in agreement with the results of the previous study by Rudi et al. (2004). Therefore, to prepare template DNA from EFSs and ESSs, we selected the prespin method because this method has only one extra step (prespin), and prior to centrifugation, it produces results similar to those from more complicated methods. All methods tested for EPSs performed equally in PCR; therefore, methods 1 (direct) or 2 (prespin method) could be used for template preparation. It is important to evaluate sensitivity of the method for the detection of pathogens in the presence of background microflora in various sample matrices (Arif et al. 2013). Using serial dilutions of Salmonella (107–101 CFU ml1) in the presence of over 109 CFU ml1 of background micro-organisms from enriched feces or soil or pecan samples, we evaluated the PCR detection sensitivity in each sample matrix, with and without AFs. Without AFs, no amplification was observed in sensitivity assays on inoculated enriched feces samples; however, a sensitivity of 40 CFU of Salmonella/reaction, similar to that of pure culture, was achieved when the AFs were added. This result agrees with the studies of Al-Soud and R adstrom 1188

(2000), who reported the addition of 04% (w/v) BSA allowed amplification by Taq polymerase in presence of a high concentration of feces per reaction (4% v/v compared to 04% of feces). Similar results were reported by Rudi et al. (2004), who demonstrated that incorporating 04% BSA in fecal samples allowed amplification of the target even in the presence of a high concentration of feces, that is, 11 mg per 50 ll compared to 25 mg per 50 ll without any inhibition in the reaction. Similarly, a sensitivity of 40 CFU of Salmonella/reaction was achieved when AFs were added to the PCR mix for testing inoculated enriched soil samples. Kreader (1996) reported elimination of PCR amplification inhibition upon incorporation of BSA in soil and other sample matrices. The combination of BSA and 1% PVP eliminated PCR inhibition when testing a variety of plant samples (Xin et al. 2003). In contrast, we found no difference in PCR sensitivity with inoculated and enriched pecan samples with or without incorporation of AFs. This finding is most likely due to lack of PCR inhibitors in pecan samples, as a detection limit of 40 CFU per reaction was readily achievable without the addition of AFs. Therefore, for pecan samples, a prespin method can be used directly without the incorporation of a PCR amplification facilitator. This developed method can deliver rapid and costeffective results than the conventional cultural methods although a lower detection limit of 1 CFU/50 g of inshell pecans has been reported by enrichment (Beuchat et al. 2012). In conclusion, the results of this study suggest that the prespin method of DNA extraction, together with the use of 04% BSA + 1% PVP in PCR, can lead to simple, rapid, cost-effective and sensitive PCR detection of Salmonella in soil, feces and in-shell pecans. Both the prespin and direct methods of DNA extraction facilitate effective PCR detection of Salmonella from surface of pecans without incorporation of AFs. This method allows large amount of different samples matrices to be processed for the detection of Salmonella without compromising the sensitivity and reproducibility of PCR assays. Acknowledgements This work was supported by the Oklahoma Agricultural Experiment Station and the Samuel Roberts Noble Foundation, Ardmore. We thank Dr. Jacqueline Fletcher and Dr. Astri Wayadande for reviewing this manuscript. We also thank Ms. Becky Cheary for providing the soil, fecal and pecan samples for the experiment. Conflict of Interest The authors declare that they have no conflict of interest.

Journal of Applied Microbiology 117, 1181--1190 © 2014 The Society for Applied Microbiology

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Salmonella detection from different matrices

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Journal of Applied Microbiology 117, 1181--1190 © 2014 The Society for Applied Microbiology

A simple, rapid, cost-effective and sensitive method for detection of Salmonella in environmental and pecan samples.

PCR is widely used in the routine detection of foodborne human pathogens; however, challenges remain in overcoming PCR inhibitors present in some samp...
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