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ENVIRONMENT, WELL-BEING, AND BEHAVIOR |
* Department of Food Engineering, Middle East Technical University, 06531, Ankara, Turkey; and Department of Biotechnology, Middle East Technical University, 06531, Ankara, Turkey
1 Corresponding author: candan{at}metu.edu.tr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: Salmonella Typhimurium • Salmonella Enteritidis • random amplified polymorphic deoxyribonucleic acid analysis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Serotype identification is necessary for investigations of foodborne outbreaks and provides the examination of the overall changes in the prevalance of a particular serotype. Salmonella Enteritidis and Salmonella Typhimurium are the 2 most common Salmonella food-poisoning serotypes in the United Kingdom (Plummer et al.,1995). It was recently reported that most of the clinical isolates of foodborne diseases from different hospitals in Ankara, Turkey, were Salmonella Typhimurium (Y
ldrmak et al., 1998). Although serotyping has shown that Salmonella Enteritidis displaced Salmonella Typhimurium as the most frequently isolated serotype, the identification of Salmonella Typhimurium serotype is still important in some countries (Eley, 1996).
Methods for the detection of Salmonella include the conventional culture method, rapid screening methods, immunological methods, and DNA-based methods (Feng, 1992). The techniques used for the detection of Salmonella mostly suffer from being either time-consuming, labor intensive, or expensive. The application of the PCR is one approach for the rapid and effective detection and identification of salmonellae (Cano et al., 1993; Way et al., 1993; Bej et al., 1994; Van Lith and Aarts, 1994). In most of these studies, PCR amplifications have been based on prior sequence information from cloned genes (Rahn et al., 1992; Aabo et al., 1993, 1995; Cohen et al., 1996). The application of random amplified polymorphic DNA (RAPD) analysis (Welsh and McClelland, 1990; Williams et al., 1990; Caetano-Anolles et al., 1991) based on the random amplification of genomic DNA fragments through short arbitrarily designed primers is an attractive alternative for the detection and identification of microorganisms (Lawrence et al., 1993; Meunier and Grimont, 1993; Cave et al., 1994; Martinez-Murcia and Rodriquez-Valera, 1994; Stephan, 1996), especially where previous sequence information is not available. Random amplified polymorphic DNA analysis has the potential to detect polymorphism throughout the entire genome as compared other techniques. It also allows one to start a blind walk through the genomic DNA of an organism and to possibly discover regions of interest that would otherwise not be easily established. Furthermore, RAPD is more suitable than other techniques, because it does not require prior knowledge of target DNA and it uses short (9 to 10 bases) primers with a small amount of starting DNA. Salmonella Typhimurium together with Salmonella Enteritidis are the most prevalent serotypes of food poisoning all over the world, and this study was performed for the development of serotype-specific primer for one of those organisms. Thus, although RAPD is mainly used for molecular typing studies of bacteria, it is selected to differentiate Salmonella Typhimurium from other Salmonella serotypes in this study. The main objective of this study was to differentiate Salmonella Typhimurium from Salmonella Enteritidis that show very similar disease symptoms and also from other Salmonella serotypes using RAPD-PCR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Salmonella and non-Salmonella species used in this work are presented in Table 1
. These bacterial strains (Yldrmak et al., 1998; 
çgen et al., 2002a) were kindly provided by the Numune Hospital Department of Bacteriology, Ankara, Turkey; Uluda
University Faculty of Medicine, Bursa, Turkey; Department of Biology, Middle East Technical University, Ankara, Turkey; Department of Food Engineering, Middle East Technical University, Ankara Turkey; Faculty of Agriculture, Department of Food Science and Technology, Ankara University, Ankara, Turkey; Refik Saydam National Hygiene Center (Refik Saydam National Culture Collection), Ankara, Turkey; Institute of Health Protection, Ankara, Turkey.
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All Salmonella strains were grown in tryptic soy broth at 37°C for 1 d. Cultures were stored on tryptic soy agar slants at 4°C. For longer periods of maintenance, bacterial strains were kept in 20% glycerol and microbank at –80°C.
Before DNA isolation, optical density measurements were performed to analyze the growth state of the cultures according to previously determined growth curves. In general, an optical density measurement of 1.4 to 2.0 at 420 nm was found to yield efficient amplifications with all the different bacterial species tested, and optical density at 420 nm = 1.9 to 2.0 was determined as optimal for the preparation of template DNA from Salmonella spp. tested. In general, Salmonella spp. reach the logarithmic phase of growth within 6 to 7 h, which is found to be the stage to give the best quality DNA for PCR amplifications. This corresponds to a concentration between 3.5 x 109 and 8 x 109 cfu/mL, as determined by serial dilutions and total plate count assay on tryptic soy agar plates.
Preparation of Template DNA for PCR
Isolation of genomic DNA was performed by the modified method of Maloy (1990). According to this method, bacterial cells from an overnight culture previously grown at 37°C in 10 mL of tryptic soy broth were transferred to 1.5-mL Eppendorf tubes and spun at 18,000 x g for 2 min. After supernatant was removed, a new 1.5-mL cell culture was added onto the pellet and again spun at 18,000 x g for 2 min. Then, the pellet was resuspended in 467 µL of Tris-EDTA buffer by repeated pipetting, 30 µL of 10% SDS, and 3 µL of 20 mg/mL of proteinase K were added, and this mixture was incubated at 37°C for 1 h. Following 1 h of incubation, an equal volume (500 µL) of phenol-chloroform-isoamylalcohol was added and mixed well by inverting the tube until the phases were completely mixed, and then the tube was spun at 18,000 x g for 2 min. The upper aqueous phase containing nucleic acids was carefully transferred to a new tube, and phenol-chloroform-isoamylalcohol extraction was repeated until the interphase was clear. The aqueous phase obtained after the last in the series of deproteinization was mixed with 0.1x volume of 3 M sodium acetate, and ethanol precipitation was performed by adding 2x volume of ice-cold 95% ethanol. The DNA was precipitated overnight at –20°C and collected by centrifugation at 25,000 x g for 15 min. Finally, the supernatant was discarded, the pellet was washed with 1 mL of ice-cold 70% ethanol to eliminate salt, and centrifuged another 30 s at 25,000 x g. The tube was allowed to air dry until all the ethanol was evaporated. The DNA was dissolved in 30 to 50 µL of Tris-EDTA buffer and stored at –20°C.
The resulting DNA concentration was determined spectrophotometrically and also by comparing the intensity of the band with that of the bands of known concentration on agarose gel.
PCR Amplifications
A 50-µL PCR mixture contained 50 ng of template DNA, 2 mM deoxynucleoside triphosphate mix, 200 pmol of primer, 2 U of Taq DNA polymerase (MBI Fermentas Vilnius, Lithuania) in 1x reaction buffer [50 mM KCl, 10 mM Tris-HCl (pH 8.8), 0.8% Nonidet P40, 1.5 mM MgCl2]. The reaction mixtures were overlaid with 30 µL of mineral oil. The PCR cycles consisted of the following steps: 1 x 90°C, 5 min; 35 x [89°C, 1 min; 32°C, 1 min; 72°C, 1.5 min]; and 1 x 50°C, 3 min. Plasmid DNA with sequence-specific primers was used as a positive control to check the proper functioning of the enzyme and other reaction components as well as the PCR machine. No DNA negative controls with one of the arbitrary primers were also performed to check for possible contaminations. Arbitrarily designed short primers 8 to 10 bases long used for PCR amplifications are shown in Table 2. The primer design considerations were the incorporation of 50 to 70% guanine-cytosine residues into the primers and the absence of palindromic sequences.
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The PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining. For routine analysis, minigels (7 cm x 6 cm) containing 0.8% agarose in 1 x TAE (0.04 M Tris acetate, 0.001 M EDTA) were prepared. For medium-sized gels (20 x 15 cm), 1% agarose in 1 x TAE was used. Gels were stained with ethidium bromide (0.8 µg/mL). Electrophoresis was performed at 70 V for 60 min. Then the gel was visualized on ultraviolet transilluminator at 320 nm. Gels were photographed by Nikon Coolpix 4500 digital photograph machine (Nikon Inc., Melville, NY).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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In our study, RAPD analysis was used to establish serotype-specific markers for Salmonella serotypes, with emphasis on Salmonella Typhimurium, due to its importance in causing the majority of Salmonella cases in Turkey (Yldrmak et al., 1998). Namely, 10 different randomly designed primers 8 to 10 bases long (Table 2
) were used in PCR in the presence of Salmonella Typhimurium DNA and DNA from other serotypes of Salmonella (data not shown). In our preliminary studies, promising results were obtained with some of these 10 primers. Primers 1, 2, and 10 gave the same amplification pattern with many Salmonella serotypes. Primers 4 and 5 did not yield any amplification products, whereas primer 8 produced 1 band of 300 bp. A unique and fairly intense amplification band of 550 bp was obtained with primer 7 for Salmonella Typhimurium and also some of the other Salmonella serotypes. Primers 6 and 9 were designed as degenerate primers with the expectation of increasing the number of bands amplified by the PCR. The results indicate that the degenerate primers indeed increase the number of amplified DNA fragments for Salmonella Typhimurium, namely, 5 to 6 bands are generated instead of 1 to 3. Accordingly, the degenerate primers 6 and 9 were not similarly effective in generating several amplification bands with all Salmonella Typhimurium. Furthermore, Salmonella Enteritidis displayed the same amplification pattern as Salmonella Typhimurium with these degenarate primers (data not shown). However, primer 3 that produced a distinguishable pattern against Salmonella Typhimurium in those preliminary studies was selected as an identification primer. This primer was tested for 8 Salmonella Typhimurium strains, which were characterized in our previous study (çgen et al., 2002b). Although primer 3 is not a degenerate primer, the amplification pattern contained more than 1 band, the size of which varied between 300 and 1,000 bp, with 2 of these bands (300 and 700 bp) being rather intense, varying from one strain to another, and the remaining being faint bands (Figure 1). In the detection of microorganisms, it is often preferable to amplify a single and intense DNA fragment that will specify the microorganism of interest. A unique and fairly intense amplification band of 550 bp was obtained with primer 7 for Salmonella Typhimurium. In this respect, primer 7 was also tested against different Salmonella serotypes in RAPD analysis. However, the results indicate that an intense DNA fragment of 550 bp is amplified from 80% of the Salmonella serotypes tested. Therefore, this primer could not act as a detection primer for all Salmonella Typhimurium tested and nor was the primer amplification product specific to genus Salmonella (data not shown). On the other hand, according to the results with primer 3, the amplification pattern of Salmonella typhimurium, with the intense 700-bp product, was found to be unique among the 8 Salmonella Typhimurium isolates (Figure 1). Additionally, 4 more Salmonella Typhimurium strains from the Institute of Pasteur in France and National Culture Type Collection in the United Kingdom were obtained from Refik Saydam National Culture Collection in Turkey and were used in RAPD-PCR with primer 3 (Figure 2) to validate results obtained for Salmonella Typhimurium isolates. They also gave same amplification bands both at about 700 and 300 bp (Figure 2).
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ldrmak et al., 1998), yet they all displayed the same amplification pattern (Table 3). Serotype Salmonella Enteritidis is a very closely related serotype with Salmonella Typhimurium. Both are pathogenic for human and animals. In this respect, identification of serotype-specific DNA marker for Salmonella Typhimurium and differentiation from Salmonella Enteritidis are very important both for human health and the food industry. Therefore, to check and express the specificity of amplification results of Salmonella Typhimurium, primer 3 was applied to DNA of different Salmonella Enteritidis isolates and other Salmonella serotypes in RAPD-PCR (Figure 3). All isolates of serotype Salmonella Enteritidis (Figure 3) and some other Salmonella serotypes (Figure 4) given in Table 1 produced a 300-bp amplification band but not a 700-bp fragment with primer 3 under the same RAPD-PCR conditions with Salmonella Typhimurium in our study. Salmonella Typhi, Salmonella Paratyphi B, Salmonella Anatum, Salmonella Strasbourg, Salmonella Orion, Salmonella Rissen, Salmonella Quakam, Escherichia coli, and Klebsiella pneumonia in RAPD-PCR with primer 3 had given neither the 300-bp band nor the 700-bp band (Table 3, Table 4). Therefore, a 300-bp band could not be specific both for Salmonella Typhimurium and for the salmonellae genus. On the other hand, specificity of the 700-bp band for Salmonella Typhimurium in our study was consistent with the homology comparing study results of McClelland et al. (2001), because they had discovered that there were non-homologous regions on DNA among these bacteria. It was thought that the specific and polymorphic 700-bp region of Salmonella Typhimurium could be located at a region that did not show homology with closely related Salmonella serotypes and Enterobacteriaceae members. Moreover, 3 of the non-Salmonella species tested indicated absence of a similar amplification pattern (Table 4). According to the results, primer 3 generated a unique and distinguishable amplification pattern with band at about 700 bp and a band at about 300 bp with Salmonella Typhimurium. Thus, a 700-bp amplification band could be used as a specific DNA marker for Salmonella Typhimurium.
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The aim of this study was to develop a rapid and easy detection and identification method for Salmonella Typhimurium for both food industry and human health and to find a specific DNA marker for this organism. A RAPD-PCR reaction, with primer 3 with 9 bp in size, was found as an effective method. All Salmonella Typhimurium isolates used in this study gave strong amplification product at about 700 bp, an additional band at about 300 bp, and rarely faint bands at 800, 900, and over 1,000 bp in size. Isolates of other Salmonella serotypes examined (Table 1) did not produce any 700-bp band, whereas some of those gave a 300-bp amplification product. Therefore, a 300-bp amplification band has not any worth as an identification marker for serotype Salmonella Typhimurium or Salmonella genus, or both, whereas a 700-bp amplification product was found as a specific polymorphic region for Salmonella Typhimurium, and this band can be used as a specific marker for differentiation of Salmonella Typhimurium from the other serotypes.
Received for publication August 23, 2007. Accepted for publication February 14, 2008.
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