evaluation of metal hydroxide immobilization and dna

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EVALUATION OF METAL HYDROXIDE IMMOBILIZATION AND DNA EXTRACTION METHODS ON DETECTION OF SALMONELLA ENTERICA FROM PORK SAUSAGE BY NESTED POLYMERASE CHAIN REACTION UDAYA SANJEEWA KUMARA RATHNAYAKA1 and SUDIP KUMAR RAKSHIT Field of Study Food Engineering and Bioprocess Technology, School of Environmental Resources and Development, Asian Institute of Technology, Klong Luang Pathumthani 12120, Thailand Accepted for Publication February 25, 2009

ABSTRACT The aim of this work was to evaluate the effectiveness of metal hydroxide immobilization and five different DNA extraction methods in detection of Salmonella enterica from pork sausage samples by nested polymerase chain reaction (PCR). Immobilization of bacterial cells by Zr(OH)4 and Ti(OH)4 was carried out prior to DNA extraction by five DNA extraction methods. The fliC gene and enterotoxin (stn) gene of Salmonella enterica was amplified by nested PCR. Both metal hydroxide immobilization and nested PCR amplification were able to increase the detection sensitivity. DNA extraction by modified Fontana and Kapperud methods were more effective compared with other methods. The amplification of enterotoxin (stn) gene provided more sensitivity of detection compared with amplification of fliC gene. This study shows that the use of metal hydroxide immobilization and nested PCR are able to increase the sensitivity of Salmonella enterica detection from meat food samples.

PRACTICAL APPLICATION In this experiment, two extraction methods were found to be more effective for the extraction of DNA from Salmonella enterica-spiked pork sausage. Immobilization of Salmonella cells by metal hydroxide and application of nested polymerase chain reaction were able to increase the detection sensitivity and reduce the possibility of false positive results. These findings are useful

1

Corresponding author. TEL: 0066870824008; FAX: 0094452280291; EMAIL: Udaya.Rathnayaka@ ait.ac.th, [email protected]

Journal of Muscle Foods 21 (2010) 801–812. © 2010, Wiley Periodicals, Inc.

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in the selection of suitable methods for rapid, sensitive and reliable detection of bacterial pathogens from meat food samples.

INTRODUCTION Salmonella is a major concern as a pathogenic microorganism in foodborne infections, causing mild to severe clinical effects (Akkaya et al. 2008a). They are gram-negative, motile, facultative anaerobic bacteria, which typically cause an intestinal infection that is accompanied by fever, abdominal cramps and diarrhea. Contaminated egg, meat and poultry products are the main sources of Salmonella infections (Wang et al. 1996; Nowak et al. 2007; Pohlman et al. 2008; Akkaya et al. 2008b). Inspection of food for the presence of Salmonella has become routine all over the world. Conventional microbiological methods used for the detection of Salmonella in foods generally require 72–96 h (Tan and Shelef 1999). Such lengthy procedures are problematic for microbial detection in food industry, especially when applied to screening large numbers of food samples. A number of rapid methods for the detection of Salmonella in foods have been developed. This includes automated detection methods (Peng and Shelef 2001), immunological methods (Smith and Desrocher 2007) and nucleic acidbased analyses (Malorny et al. 2007). However, there are still problems with the sensitivity and specificity of those methods (Jenikova et al. 2000). Polymerase chain reaction (PCR)-based techniques are the most popular nucleic acid-based techniques. These techniques are powerful diagnostic tools for the analysis of microbial infections as well as microorganisms present in food samples (Malorny et al. 2003). The PCR is a sensitive, rapid technique, in which a few copies of target DNA can be amplified to a level detectable by gel electrophoresis. The capacity of PCR to detect microorganisms depends on the purity of the template used as target and on the presence of a sufficient number of target molecules (Estrada et al. 2007). The presence of PCR inhibitors in food samples and the minimum cell requirement are some of the limitations in PCR-based assays that lead to false negative results. The removal of inhibitory substances and rapid and efficient DNA extraction in the preparation of samples for PCR-based detection of food pathogens is important (Jenikova et al. 2000). So, the application of PCR-based methods is closely linked to the selection of suitable methods for DNA extraction (Amagliani et al. 2007). The present study evaluates the efficiency of metal hydroxide immobilization as a means of concentrating dilute samples, five different techniques used for DNA extraction from Salmonella enterica spiked in pork samples and

DETECTION OF SALMONELLA ENTERICA BY NESTED PCR

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their detection by nested PCR. These procedures were expected to increase the sensitivity and reduce the possibility of false positive results.

MATERIALS AND METHODS Bacterial Inoculum Preparation Salmonella enterica was cultured using tryptic soy broth yeast extract (TSBYE) medium, which contains 30 g of tryptic soy broth powder with dextrose, 6 g of yeast extracts and 1 L of water. Cultures were incubated 4 h at 36C to mid-exponential growth phase and serially diluted (101–1010 cfu/mL) in sterile distilled water for enumeration. Bacteria were enumerated using Rambach agar plates at 37C overnight, and bacterial concentration was estimated by calculating the average number of red colonies on plates containing 30–300 colonies. Bacterial dilutions contained 10°–103 cfu/mL were prepared using the same mid-exponential phase bacterial culture. Five grams of pork sausage were inoculated by 1 mL of each dilution, placed in 45 mL of TSBYE medium and homogenized using a stomacher for 90 s. Then the cultures were enriched for 4 h at 37C and used for DNA extraction followed by nested PCR. DNA Extraction Enriched bacterial cultures were used for DNA extraction by five methods with and without metal hydroxide immobilization with three replicates. The method proposed by Lacore et al. (2000) was used with some modifications for immobilization of bacteria with zirconium hydroxide and titanium hydroxide. One milliliter of culture was added to 2 mL of 0.324 M metal hydroxide, agitated at room temperature, vortexed and centrifuged at 12,500 rpm for 5 min at 7C. After centrifugation the pellet was obtained and used for DNA extraction by each extraction method. For nonimmobilized samples, 1 mL of the dilution series was centrifuged at 12,500 rpm for 5 min at 7C to obtain bacterial pellets. The first extraction protocol was a method developed by Fontana et al. (2005) and modified by Estrada et al. (2007). The pellets obtained by centrifugation of metal hydroxide immobilized and nonimmobilized bacterial dilutions were washed with 200 mL of ammonia hydroxide, 200 mL of absolute ethanol, 400 mL of petrol ether and 20 mL of SDS (10%).The sample was then centrifuged at 12,500 rpm for 10 min at 4C, and the pellet was resuspended in a solution containing 200 mL of 6M urea, 200 mL of absolute ethanol, 400 mL of petrol ether, 80 mL of SDS (10%) and 13 mL of 3 M sodium acetate. A second centrifugation for 10 min at 12,500rpm at 4C was also performed, and the pellet

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was resuspended with 600 mL of TE buffer (Tris–ethylene diamine tetra-acetic acid [EDTA]) pH 8.0, 35 mL of sodium dodecyl sulfate (SDS) (10%) and 10 mL of DNase-free RNase (10 mg/mL).The tubes were incubated at 37C for 30 min before the addition of 10 mL of proteinase K. This preparation was incubated at 37C for 30 min. Finally, 130 mL of 6 M sodium perchlorate and 500 mL of phenol chloroform isoamyl alcohol (25:24:1; pH 6.7) were added for DNA extraction. The tubes were then centrifuged at 12,500 rpm for 5 min, the aqueous phase was collected and the nucleic acids were precipitated with absolute alcohol. DNA was dissolved in 20 mL of TE buffer. The second DNA extraction protocol was a method developed by Kapperud et al. (1993) and modified by Estrada et al. (2007). The pellets obtained by centrifugation of metal hydroxide immobilized and nonimmobilized bacterial dilutions were resuspended in 50 mL of 1X PCR buffer containing 0.2 mg of proteinase K/mL. After being incubated at 37C for 1 h, the suspension was boiled for 10 min and then centrifuged at 12,500 rpm for 5 min at 4C. The supernatant was used for performing the PCR. The third protocol was Triton X-100 method. The pellets obtained by centrifugation of metal hydroxide immobilized and nonimmobilized bacterial dilutions were diluted 1:10 with 1–2% Triton X-100 and vortex. The suspension was heated in boiling water for 10 min and the tubes were cool to room temperature. Equal volume of chloroform was added to suspension and mixed well. The suspension was centrifuged at 12,500 rpm, 10 min at room temperature. To the upper phase equal volume of 100% isopropyl alcohol was added and mixed gently by inverting the tube and then centrifuged for 10 min at 12,500 rpm, 7C. The DNA pellet obtained was washed with 1 mL of 70% ethanol, centrifuged at 12,500 rpm, 5 min, 7C and freeze-dried. Then dissolve in 20 mL of TE buffer and store at -20C until use. The fourth extraction method was a method proposed by Bansal et al. (1996). The pellets obtained by centrifugation of metal hydroxide immobilized and nonimmobilized bacterial dilutions were washed once with 1 mL of pH 7.4 phosphate buffered saline, and resuspended in equal volume of cold water and incubated in a boiling water bath for 10 min. The clear supernatants obtained after a 5 min of centrifugation at 12,500 rpm were stored at -20C and used for PCR. The fifth extraction method was the protocol proposed by Sambrook and Russel (2001). The pellets obtained by centrifugation of metal hydroxide immobilized and nonimmobilized bacterial dilutions were resuspended in 0.5 mL of lysis solution (8 M urea, 0.3 M NaCl, 10 mm Tris–HCl) with the addition of 0.5 mL 10% SDS. After 20 min at 37C, DNA was extracted with two volumes of phenol by mixing in 10 min and centrifuging at 12,500 rpm for 10 min. An equal volume of chloroform : isoamyl alcohol was added to the aqueous phase and then samples were mixed as above and centrifuged at


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12,500 rpm for 5 min. The nucleic acids in the aqueous phase were then precipitated with 2.5 volumes of ethanol and 1/10 volume of 3 M sodium acetate (pH 5.2) by incubating at -20C for 1 h and centrifugation. DNA pellets were washed with 70% ethanol and dissolved in 20 mL of TE buffer and store at -20C until use. Determination of DNA Quality and Quantity The quality of the extracted DNA by all methods was investigated by measuring absorbance at 260 and 280 nm using spectrophotometer and calculating the A260/A280 ratios. DNA yield of extracted DNA was also measured using spectrophotometric readings and assuming that 1 OD at 260 nm equals 50 mg/mL DNA (Sambrook et al. 1989). Nested PCR The external and internal primers located on the conserved regions of the fliC gene, which was identified by Touron et al. (2005), was used for nested PCR. Those primers are 5′-CGGCGTGAAAGTCCTGGCG-3′ (Sal345), 5′-CGGTGTTAAAGTCCTGTCT-3′ (Sal345d), 5′-CGAATCTTCGATACGG CTACG-3′ (Sal1312), 5′ CATCTTCGATACGGCTACG-3′ (Sal1312d), 5′-AT CCAGGTTGGTGCTAACG-3′ (Salnes3d), 5′-TTTACGGTTTGCCCAGG-3′ (Salnes1), 5′-CCGTATTGCCAAGGTTGG-3′ (Salnes1d). 1 mL of DNA extracted by different extraction methods were used for PCR amplification in a 50 mL final volume of the following mixture: 25 mL of PerfectShotTM Ex Taq (Takara, Shiga, Japan), 0.25 mM of each external primer (Sal345, Sal345d, Sal1312, Sal1312d), and sterilized distilled water. PCR amplification was performed with 10 min at 95C for denaturation, followed by a 35-cycle program (denaturation at 95C for 1 min, annealing at 55C for 1 min, extension at 72C for 1 min), and a final extension step at 72C for 10 min. The nested PCR was processed using a 1 mL volume of the previous PCR products following the same reaction mixture but with internal primer concentrations of 0.25 mM of each internal primer: (1) Salnes1; (2) Salnes1d; and (3) 0.5 mM of the internal primer Salnes3d. Nested PCR amplification was performed with the same PCR protocol. The amplified DNA products were analyzed by gel electrophoresis in 2.5% agarose gel containing ethidium bromide in Tris base, acetic acid and EDTA buffer (20 mm Tris acetate, 0.5 mm EDTA, pH 7.8). PCR products were visualized by UV illumination and their images were recorded. Four pairs of PCR primers designed by Ul-hassan et al. (2004) for enterotoxin (stn) gene were also used to test the efficiency of different DNA extraction methods by a nested PCR run. First step PCR was carried out with the primers QVR137, 5′-GGTCAAAATCCAGCGGTTTA-3′ and QVR138, 5′-TTGCTGCTAACGGCGAGA-3′. The second round of PCR was carried

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out with primers QVR139, 5′-GCCGGCTTTCAACGCCTCTAC-3′ and QVR140, 5′-GACCAAAGCTGACGGGACAG-3′. The same PCR conditions and procedure used above were followed. RESULTS AND DISCUSSION Detection of Salmonella in meat foods has involved culturing techniques (Hara-kudo et al. 2001), immunological test (Wang et al. 1996) and nucleic acid-based assays (Malorny et al. 2007). Comparing to those methods, PCRbased methods have been recognized to be more sensitive, specific and rapid for food pathogen detection. Effective way to acquire bacterial cells from the food matrix, effective DNA extraction methods and minimum number of cells required (sensitivity) are the major concerns in improvement of detection sensitivity of PCR-based techniques. When applying this method for food products, bacterial cells have been acquired and concentrated from food matrix prior to DNA extraction, using immunomagnetic separation (Jenikova et al. 2000) and metal hydroxide immobilization (Lacore et al. 2000). Although immunomagnetic separation is more specific, metal hydroxide immobilization is cheaper and easier. The quantity and purity of extracted nucleic acid are important factors in PCR-based detections. Selection of a proper method to extract appropriate amount of DNA containing minimum level of proteins, RNA or any other PCR inhibitors determines the success and the validity of the PCR analysis (Amagliani et al. 2007). Other important factors that should be taken in to account when selecting a DNA extraction method are: the time need to complete the extraction and the toxicity and expenses of the chemical product employed in extraction (Chapela et al. 2007). Quality and Quantity of Extracted DNA Purity of the extracted DNA was in the acceptable range (1.6–1.8) for all extraction methods (Table 1). Immobilization with metal hydroxide increased the DNA yield of all the methods tested. DNA yield using Zr(OH)4 immobilization was higher than Ti(OH)4 immobilization. Highest DNA yield was obtained by the modified Fontana et al. (2005) method for the sample immobilized with Zr(OH)4 and lowest yield from the Bansal et al. (1996) extraction method for nonimmobilized samples. Based on the results, Zr(OH)4 seems to be better than Ti(OH)4 for immobilization of Salmonella. Nested PCR DNA was extracted by five extraction methods with and without metal hydroxide immobilization from enriched cultures obtained by initial inocula-


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TABLE 1. QUANTITY AND PURITY OF EXTRACTED DNA BY DIFFERENT EXTRACTION METHODS Extraction method

Immobilization

DNA content (mg)

DNA purity (A260/A280)

Modified Fontana et al. (2005) method

Immobilized with Zr(OH)4 Immobilized with Ti(OH)4 Nonimmobilized

33.5 ⫾ 0.02 28.9 ⫾ 0.26 17.8 ⫾ 0.30

1.64 ⫾ 0.02 1.63 ⫾ 0.01 1.64 ⫾ 0.02

Modified Kapperud et al. (1993) method


25.8 ⫾ 0.56 19.1 ⫾ 0.26 15.6 ⫾ 0.10

1.67 ⫾ 0.02 1.64 ⫾ 0.01 1.64 ⫾ 0.01

Triton X-100 method


20.2 ⫾ 0.26 16.5 ⫾ 0.10 14.8 ⫾ 0.46

1.69 ⫾ 0.02 1.69 ⫾ 0.01 1.77 ⫾ 0.03

Bansal et al. (1996) method


11.4 ⫾ 0.26 8.7 ⫾ 0.40 8.9 ⫾ 0.26

1.51 ⫾ 0.02 1.45 ⫾ 0.01 1.45 ⫾ 0.02

Sambrook and Russel (2001) method


15.4 ⫾ 0.17 13.7 ⫾ 0.17 11.2 ⫾ 0.20

1.62 ⫾ 0.02 1.64 ⫾ 0.02 1.64 ⫾ 0.01

tion of 103–10° cfu/mL in TSBYE medium with three replicates. The extracted DNA was then amplified using the nested PCR. In gel electrophoresis, same results were observed for all replicates in each treatment. The sensitivity of detection was increased by metal hydroxide immobilization for amplification of both fliC and enterotoxin (stn) genes. Immobilization with Zr(OH)4 was more successful compared with immobilization with Ti(OH)4 in sensitivity increase. The detection of the amplification of fliC gene by external primers for nonimmobilized samples required a minimum of 103 cfu/mL for all extraction methods tested (Table 2). Immobilization with Zr(OH)4 enabled to increase the detection sensitivity of amplification by external primers up to 102 cfu/mL for modified Fontana et al. (2005) method, modified Kapperud et al. (1993) method and Triton X-100 method. Immobilization with Ti(OH)4 increased the detection sensitivity of amplification by external primers up to102 cfu/mL for the DNA extracted by modified Fontana et al. (2005) method. The second step PCR with internal primers for fliC gene increased the sensitivity of all methods except the method proposed by Sambrook and Russel (2001) when used for nonimmobilized samples. The detection sensitivity of the modified Fontana et al. (2005) method was increased for both immobilized sample with Zr(OH)4 and nonimmobilized sample by 100-fold. Similar results were obtained using the modified Kapperud et al. (1993) method for extraction without immobilization. The second step PCR increased the sensitivity by 10-fold for all other methods tested. Besides specificity, the nested PCR increased the sensitivity as well.

With Zr(OH)4 With Ti(OH)4 Nonimmobilized





Modified Fontana et al. (2005) method

Modified Kapperud et al. (1993) method

Triton X-100 method

Bansal et al. (1996) method

Sambrook and Russel (2001) method

-

+ + + + + +

-

-

-

-

-

-

+ + -

+ + +

+ + +

+ + +

-

-

-

+ +

-

-

-

-

+ -

+ -

-

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

-

-

+ + -

+ + -

+ + -

-

-

-

+ -

+ -

-

-

-

-

-

100

+ + +

-

+ + +

101

+ -

-

102

+ + +

-

103

+ + -

100

+ + +

101

102

102

103

100

PCR first step

Nested PCR

PCR first step 101

Enterotoxin (stn) gene

fliC gene

103, 102, 101 and 10° are bacterial concentrations cfu/mL. + and - are presence and absence of bands in gel electrophoresis of PCR products, respectively.

Immobilization methods

Extraction methods

TABLE 2. NESTED PCR AMPLIFICATION RESULTS OF fliC GENE AND ENTEROTOXIN (stn) GENE

+ + +

+ + +

+ + +

+ + +

+ + +

102

+ -

+ -

+ + -

+ + -

+ + -

101

Nested PCR

-

-

-

+ -

+ -

100

808 U.S.K. RATHNAYAKA and S.K. RAKSHIT


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The minimum cell requirement for detecting amplification of enterotoxin (stn) gene by primers QVR137 and QVR138 for nonimmobilized samples was 103 cfu/mL for all extraction methods tested (Table 2). Immobilization with Zr(OH)4 enabled the increase of detection sensitivity of amplification by the same primers up to 101 cfu/mL for the DNA extracted by the modified Fontana et al. (2005) and Kapperud et al. (1993) methods. The detection sensitivity was increased up to 102 cfu/mL for the DNA extracted by the Triton X-100 method. Immobilization with Ti(OH)4 increased the sensitivity of amplification by the same primers up to102 cfu/mL for the DNA extracted by modified Fontana et al. (2005), Kapperud et al. (1993) and Triton X-100 methods. The second step PCR with primers QVR139 and QVR140 increased the detection sensitivity of all methods tested. The detection sensitivity of the Bansal et al. (1996) and Sambrook and Russel (2001) extraction methods were increased by 100-fold for the samples immobilized with Zr(OH)4. For all other methods the sensitivity was increased by 10-fold by the second step of PCR (Table 2). The amplification of enterotoxin (stn) gene provided more sensitivity of detection compared with amplification of fliC gene. The modified Fontana et al. (2005) and Kapperud et al. (1993) extraction methods tested in this experiment are found to be the best methods that could be used in routine detection of Salmonella enterica from pork meat samples. The combined application of metal hydroxide immobilization prior to DNA extraction and nested PCR were able to increase the sensitivity of detection to 1 cfu/mL using the two extraction methods. The other three extraction methods tested were able to give 101 cfu/mL sensitivity with Zr(OH)4 immobilization and nested PCR for enterotoxin (stn) gene. Compared with those methods, the Bansal et al. (1996) method is simple, requires less harmful chemicals and is less time consuming. Nested PCR of DNA extracted by this method with metal hydroxide immobilization could be applied for preliminary testing of samples with high possibility to be positive for Salmonella. The enrichment step, which was reduced to 4 h in this experiment compared with overnight enrichment practice to obtain detectable number of cells, could decrease the overall experimental time to one working day compared with two working days of reported nested PCR applications (Amagliani et al. 2007) and 7–10 days of standard conventional microbiology methods. With these results it can be concluded that the combined application of metal hydroxide immobilization and nested PCR is a powerful and sensitive method for the detection of Salmonella in meat food samples. The Bansal et al. (1996) method is the best method for preliminary studies for high-risk samples because of its simplicity of use and suitability for samples with high number of pathogens. Modified Fontana et al. (2005) and Kapperud et al. (1993) methods are highly sensitive for PCR detection especially if nested PCR is carried out. The overall outcomes of this study are easy separation and con-

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centration of Salmonella cells from meat food samples using Zr(OH)4 immobilization, suitable DNA extraction method, less enrichment time and high sensitivity using nested PCR method. REFERENCES AKKAYA, L., ALISARLI, M., CETINKAYA, Z., KARA, R. and TELLI, R. 2008a. Occurrence of Escherichia coli O157:H7/O157, Listeria monocytogenes and Salmonella spp. in beef slaughterhouse environments, equipment and workers. J. Muscle Foods 19(3), 261–274. AKKAYA, L., CETINKAYA, Z., ALISARLI, M., TELLI, R. and GOK, V. 2008b. The prevalence of E. coli O157/O157:H7, L. monocytogenes and Salmonella spp. on bovine carcasses in Turkey. J. Muscle Foods 19(4), 420–429. AMAGLIANI, G., GIAMMARINI, C., OMICCIOLI, E., BRANDI, G. and MAGNANI, M. 2007. Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods. Food Control 18, 1137–1142. BANSAL, N.S., MCDONELL, F.H., SMITH, A., ARNOLD, G. and IBRAHIM, G.F. 1996. Multiplex PCR assay for the routine detection of Listeria in food. Int. J. Food Microbiol. 33, 293–300. CHAPELA, M.J., SOTELO, C.G., PEREZ-MARTIN, R.I., PARDO, M.A., PEREZ-VILLAREAL, B., GILARDI, P. and RIESE, J. 2007. Comparison of DNA extraction methods from muscle of canned tuna for species identification. Food Control 18, 1211–1215. ESTRADA, C.S.M.L., VELASQUEZ, L.D.C., GENARO, S.D. and GUZMÁN, A.M.S.D. 2007. Comparison of DNA extraction methods for pathogenic Yersinia enterocolitica detection from meat food by nested PCR. Food Res. Int. 40, 637–642. FONTANA, C., COCCONCELLI, P.S. and VIGNOLO, G. 2005. Monitoring the bacterial population dynamics during fermentation of artisanal Argentinean sausages. Int. J. Food Microbiol. 103, 131–142. HARA-KUDO, Y., KUMAGAI, S., MASUDA, T., GOTO, K., OHTSUKA, K., MASAKI, H., TANAKA, H., TANNO, K., MIYAHARA, M. and KONUMA, H. 2001. Detection of Salmonella enteritidis in shell and liquid eggs using enrichment and plating. Int. J. Food Microbiol. 64, 395–399. JENIKOVA, G., PAZLAROVA, J. and DEMNEROV, K. 2000. Detection of salmonella in food samples by the combination of immunomagnetic separation and PCR assay. Int. Microbiol. 3, 225–229. KAPPERUD, G., VARDNUND, T., SKJERVE, E., HORNES, E. and MICHAELSEN, T.E. 1993. Detection of pathogenic Yersinia entero-


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