0000013441 00000 n Ashton, W. L. G. (1990). They would like to acknowledge Ghariani Mohamed for his assistance with laboratory testing of samples and also, Chaker Lilia for coordinating this study, Regional laboratory of Hygiene Tertiary Care Hedi-Chaker, Sfax, Tunisia. Indeed, geographical and climatic differences may also contribute to these differences (Wang et al., 2013; Yang et al., 2013; Dong et al., 2014). <> After inoculation, samples were stomached for 45 s. One milliliter of sample was subjected to the DNA extraction using the method mentioned above. /Contents 18 0 R>> Almeida, C., Cerqueira, L., Azevedo, N. F., and Vieira, M. J. 32 0 obj 18 0 obj hb```2 NE! >00(\UY*Z"R=q1!^dn8irESKK[qTJsQk{KD^e) @NQRALJ @a47} 09wZ'120%1N0:i:n31Zm9X)/.j f 3,* 57f Immunol. startxref x_7*SO@\.Nsl@SO?08b FkVw6/!xQ[qbAC7)G]ULG`Dzw9IpawAr* uLt=i_F*jMwpaawi*4Nr@#& +r Polymerase chain reaction (PCR) assay demonstrated the specificity of invA primers for detection of Salmonella as confirmed by biochemical and serological assay. HlTn1+s#Oe$0 InBHlHnW5sg,)9Oc/g1wI.Udi}a]JIhtXsI$Oku1{w[ 9yyO+J-m 7k}haFf:xDKB|LaB8@!`9fVXisCvr#{]>m.1 {pN8. (2008). Biol. <> endstream The qPCR assay exhibited a good intra and inter-run reproducibility. My-IAC probe was labeled at the 5- end with Cy5 dyes as reporter and at the 3- end with Iowa Black RQ-Sp as quencher. Food Microbiol. endobj Indeed, qPCR and extraction controls consistently yielded negative results confirming that there are no false-positivent findings and the invA amplification is derived solely from food associated Salmonella spp. Primer sequences for virulence genes in Salmonella spp. However, this serotype was not detected in our range of food. Web9.4.2 Procedure for food, animal feed samples, and environmental samples Detection of Salmonella Typhi and Salmonella Paratyphi is described in Annex D. 38 0 obj Methods 70, 245251. <> in pig mesenteric lymph nodes (MLN), but there are no published estimates of the diagnostic The data were obtained from the study. On the other hand, Webber et al. Marcus S. L., Brumell J. H., Pfeifer C. G., Finlay B. DNA in raw poultry meat 27/45 (60%), milk 31/93 (33.3%), raw red meat 5/13 (38.5%), and fish 11/46 (23.9%) samples. Cell Biol. Gel image of the PCR amplification of a DNA fragment from the invA gene (284bp) of representative Salmonella strains isolated from broiler farms. <> Sacristan S., Goss E. M., Eves-Van Den Akker S. How do pathogens evolve novel virulence activities? Indeed, a positive result at the lowest tested contamination level of Salmonella spp. WebSalmonella, in which a specific bacteriophage was engineered to carry a detectable marker (ice nucleation gene). doi: 10.1016/j.meatsci.2016.04.037. Salmonella spp. International microbiology : the official journal of the Spanish Society for Microbiology, A combination of immunomagnetic separation and polymerase chain reaction (IMS-PCR) was used to detect Salmonella in food samples. Likewise, the presence of the csgA gene is relevant to public health because the csg genes in Salmonella are related to the ability to produce biofilms, leading to increased drug resistance [37]. endobj endobj Appl. 186, 613. endobj Microbiol. (2013) showed fairly significant levels of contamination of broilers with S. Enteritidis and S. Kentucky but they indicated that London and Irena serovars are the most isolated from clam, i.e., sea food but no S. Kentucky contamination. Enumeration of Salmonella bacteria in food and feed samples by real-time PCR for quantitative microbial risk assessment. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. The pattern III was the most predominant that was detected in 10 isolates (Heidelberg 3, Paratyphi B 4, Enteritidis 2). An overview of detection methods for various foodborne pathogens, including Listeria monocytogenes, Salmonella enterica, and shiga toxin-producing Escherichia coli, and bacterial toxins in food matrices, with emphasis on those methods which do not require cultural enrichment. Primers and annealing temperature used for PCR are listed in Table 1. This is a summary of the ISO 6579 Part 1: Detection of Salmonella spp. qPCR showed a specific amplification corresponding to Salmonella spp. endstream endobj 224 0 obj <> endobj 225 0 obj <>stream WebFoodborne diseases are an important cause of morbidity and mortality. To be in line with the performance characteristics determined for detection of Salmonella spp. endstream The serotype, infective dose, virulence factors, and host immunity will influence the disease's clinical presentation [6]. Abdullahi U. F., Igwenagu E., Muazu A., Aliyu S., Umar M. I. Intrigues of biofilm: a perspective in veterinary medicine. reported that 88.9% (iroN; 112/126) and 79.4% (sitC; 100/126) of the Salmonella Heidelberg carried the gene [25]. The different food samples were transported under complete aseptic conditions in an icebox within 2 h to the regional hygiene care laboratory of Sfax (Southeast of Tunisia) for processing. Briefly, a total of 25 g or 25 mL of sample was homogenized with 225 mL of Buffered Peptone Water (BPW) (Oxoid, Basingstoke, United Kingdom), stomached for 45 s and followed by incubation at 37C for 22 h. After pre-enrichment, 100 l and 1 mL samples were taken and mixed with 10 ml of Rappaport Vassialidis soya (RVS) broth (BD Difco, Germany) and Muller Kauffmann tetrathionate-novobiocin (MKTTn) broth (biokar, France), respectively. doi: 10.1016/j.fm.2016.03.005, Kasturi, K. N., and Drgon, T. (2017). 0000008536 00000 n Three different stool sample-processing methods (centrifugation, immunomagnetic separation, and selective enrichment cultivation) for the identification of Salmonella serogroups by PCR were studied and the PCR assay with overnight enrichment performed as well as, or even better than, the conventional culture technique. 1. Thus, fluorescent-probe-based TaqMan real-time PCR (qPCR) systems have been developed in the last decade to detect Salmonella nucleic acids in food samples. 57, 195203. Each dilution was tested in duplicate. J. Microbiol. For this reason, it is possible that the presence of the sefA gene in Salmonella Heidelberg isolates could indicate a major virulence of the strains. endobj J. Electrochem. {"type":"entrez-nucleotide","attrs":{"text":"M90846.1","term_id":"154154","term_text":"M90846.1"}}. System PCR Assays for. stream J.N.Hdjch!YB.8{2H{4>i[29d 1 $slZTnk@mJYvgp*"d~a*S SZZG !\^^jj|A ZFZ!%(QnX!0SP4RR P\N@ZFT f18`g0=;anL84$ X32e:`R1LL3 0ar4`Me^ F' cD^l0 Ryi2}o7+x]WY=&)-h6ff9OjwI1\ &sF!*gUs"m)qx6/. Food Microbiol. Federal government websites often end in .gov or .mil. WebSalmonella in foods: Detection (AOAC, 1995m). /Length 6481 >> L!5=\,)eZ=_:~ny5*mS|Z Accordingly, the results of this study could lay the foundation for further research on public health security and food safety problems caused by Salmonella infections in Colombia. hb```b``f`c`u`f@ a;GMUt}& Sq0+0yA*JT%"CS&3 *BFx9D Detection of the twenty-four virulence genes by PCR classified 49 selected Salmonella isolates into 21 patterns (Table 2). DNA template for qPCR was extracted from an overnight pre-enriched sample in buffered peptone water using lysisguanidine isothiocyanate method. Microbiol. Grimont, P. A. D., and Weill, F. X. A pre-enrichment step was not included in this experiment. 25 0 obj endstream The optimal qPCR efficacy was achieved using cycling profile including a denaturation at 95C for 30 s, followed by 40 cycles of 05 s at 95C and 30 s at 60.0C. Evaluation of corn oil as an additive in the pre-enrichment step to increase recovery of Salmonella enterica from oregano. However, the spvB gene that is present in these isolates is relevant because spv genes are highly associated with strains that cause nontyphoid bacteremia and disseminated infection in humans [17, 65]. <>>>] WebThis chapter provides recommendations on the prevention, detection and control of Salmonella in poultry. Vet. 43 0 obj <> The reactions were carried out following the manufacturer's recommendation for the GoTaq Flexi DNA Taq polymerase (Promega, Madison, WI, United States), 1L of DNA, and 1L of each primer (10pmol/L). endobj 167 - 172 Article Download PDF Google Scholar 12 Diferentes mtodos para aislamiento y deteccin de Salmonella spp. in samples from the primary production stage, LOD 50 values for detection of Salmonella in food samples were calculated from the raw data of the validation studies performed in 2000. (2007). x3R235W(*T0PR0T(Y@@QC= P AJ 14PpW stream stream Our study is the first to use a combined enrichment/invA qPCR method as a screening assay to detect Salmonella DNA in different food matrices in Southern Tunisia. The probe was labeled with a fluorescent reporter dye, 6-carboxyfluorescein (FAM), on the 5-end and with Black Hole Quencher (BHQ) at the 3 end of the probe. <> Control. x3R235W(*T0PR0T(Y@@QC= P AJ 14SpW Webautomated in-vitro diagnostic test designed for qualitative detection of Salmonella, Campylobacter and Shigella/enteroinvasive Escherichia coli (EIEC) in human faecal samples from patients with signs and symptoms of gastrointestinal infection. Materials and Methods One hundred and Fifty food samples of meat, Rapid detection and discrimination of pathogenic stream <> <> endobj Hierarchical effector protein transport by the Salmonella typhimurium SPI-1 Type III secretion system. 0000012210 00000 n endobj 74, 12991304. WebView PDF. 0000004269 00000 n Wolffs, P. F., Glencross, K., Thibaudeau, R., and Griffiths, M. W. (2006). endstream endobj 215 0 obj <>>> endobj 216 0 obj >/PageUIDList<0 23757>>/PageWidthList<0 612.0>>>>>>/Resources<>/ExtGState<>/Font<>/ProcSet[/PDF/Text/ImageC]/XObject<>>>/Rotate 0/Tabs/W/Thumb 192 0 R/TrimBox[0.0 0.0 612.0 792.0]/Type/Page>> endobj 217 0 obj <> endobj 218 0 obj <> endobj 219 0 obj [/ICCBased 239 0 R] endobj 220 0 obj <> endobj 221 0 obj <> endobj 222 0 obj <> endobj 223 0 obj <>stream Rapid detection of foodborne pathogens with high sensitivity and specificity is becoming an urgent requirement in health safety, medical diagnostics, environmental safety, and controlling food quality. Available at: https://www.iso.org/obp/ui/#iso [accessed May 2, 2016]. isolated from poultry: a global overview. The precise number of CFU was confirmed using the plate count method onto nutritive agar (Oxoid, Basingstoke, United Kingdom). stream {e?uC~7~7~7~7~76\X_X_9TTOe1]53 8HGJ:yc:q>p:u}8A[ C= Castro-Var R., Rubio L. F. d., Vega A., Rondon-Bar I. Phenotypic and genotypic resistance of Salmonella Heidelberg isolated from one of the largest poultry production region from Colombia. Schrader, C., Schelke, A., Ellerbroek, L., and Johne, R. (2012). Also, the sitC gene is another gene that is related to iron metabolism, and this gene encodes an important transporter of iron [67], and all the isolates carried the gene. WebThe detection of Salmonella in animal faeces and in samples of the primary production stage necessitates four stages, as described in Ch. However, similar studies should be performed for any food matrices since they differed in their organic compounds. The (my-IAC) heterologous DNA sequence, primers, and probe used in this work were described previously (Deer et al., 2010) and were kindly provided by Narjol Gonzalez Escalona (FDA, CFSAN). <> Castro-Vargas R. E., Herrera-Snchez M. P., Rodrguez-Hernndez R., Rondn-Barragn I. S. Antibiotic resistance in Salmonella spp. (2008). In the same way, previous research has detected sipB, orgA, prgH, and spaN genes in Salmonella isolates from poultry-related sources [47]. Before 0000011651 00000 n Previous studies of virulence gene detection in Salmonella Heidelberg isolated from chicken carcasses did not report isolates with the sefA gene [25]. stream <> The sopE gene was present in 70% (n=7/10) of the human isolates, respectively. The lowest inoculated detection limits for Campylobacter and Salmonella species in artificially contaminated chicken meat samples were 10 3 CFU and 1 CFU per 25 grams, respectively, after 72, 38963900. S. Enteritidis, the most isolated serotype in chicken industry and food poisoning was also not found in our samples. (2010). Isolation and Identification of Salmonella from Meat, Poultry, x3R235W(*T0PR0T(Y@@QC= P AJ 1QpW Table 1 shows Salmonella spp. The presence of 24 virulence genes was evaluated using end-point PCR. They were blasted against the nucleotide database of the NCBI website2 to ensure identity among reported BLAST sequences for the target gene and the absence of significant homology with other microorganism sequences. Salmonella strains used for specificity testing. Among the 25 Salmonella isolates, a total of eight serotypes were identified. x In humans, the consumption of chicken meat and eggs that were contaminated is conduced to develop the disease because they are considered the main reservoir and vehicle of Salmonella [1, 4]. 24 0 obj For example, PCR-based methods can be employed for the detection of pathogenic E. coli and Salmonella in beef [ 28 ]. /Contents 46 0 R>> National Library of Medicine endobj Laboratory Protocol Isolation of Salmonella spp. And Weill, F. X nutritive agar ( Oxoid, Basingstoke, United Kingdom ) CFU was confirmed the. [ 6 ] III was the most predominant that was detected in our samples Salmonella by. 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