Stock solution of food samples contained many types of bacteria differentiated with the colors shown in Figure 1. Winnen B., Schlumberger M. C., Sturm A., et al. Webdifferential agar. Xia Y., Li H., Shen Y. Antimicrobial drug resistance in Salmonella enteritidis isolated from edible snakes with pneumonia and its pathogenicity in chickens. /Contents 20 0 R>> Each qPCR included one positive control (S. enterica DNA), no template control (mastermix and sterile water) and a negative DNA control (Escherichia coli DNA). WebThe results support the adequacy of the ISO for the detection of Salmonella spp. Influence of enrichment media and application of a PCR based method to detect Salmonella in poultry industry products and clinical samples. WebThe concentration and detection of Salmonella by TFF for surface water samples spiked with a ve-strain cock-tail of Salmonella are shown in Table 1. Shen Y., Xu L., Li Y. Biosensors for rapid detection of. endobj and Listeria spp., and to give information about species and March 2018 - Added real-time quantitative PCR for confirmation of Salmonella isolates protocol and validated preenrichment changes for leafy produce, hbbd``b`
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11 0 obj The qPCR showed a consistent detection of two copies of the invA gene/PCR reaction, a good intra- and inter-run reproducibility with a good PCR efficiency (89.6%). Virulence gene distribution of Salmonella pullorum isolates recovered from chickens in China. reviewed and edited the manuscript; M.P.H.-S., K.J.L.-V., and I.S.R.-B. 132, 365386. stream WebThe sample preparation and microarray method used in this study increased sensitivity and reduces time-to-result for detection of Salmonella. J. Food processors and associated laboratories around the world use the BAX System for accurate, reliable Salmonella detection in raw ingredients, finished products and environmental samples. The analytical sensitivity of qPCR using a 10-fold serially diluted plasmid in DNase- and RNase-free water were analyzed in a range from 2.00 100 to 2.00 105 gene copies per PCR reaction. For PCR-based patterns, black area represents a positive result and white area represents negative result for the presence of a virulence gene. Food Microbiol. Data from 25 out of the 500 tested food samples were in agreement by both methods giving a relative sensitivity of 100% for our qPCR. <> 16-h magnetic immuno-PCR assay can be promising for routine use in the detection of Salmonella in chicken meat samples, and it consequently may prevent the risk ofSalmonella infections in regard to chicken meats. Antigenic Formulae of the Salmonella Serovars, 9th Edn. Food sources of Salmonella included mainly milk, eggs, meat (poultry, beef) vegetables, and fresh fruits (Almeida et al., 2013). WebAmong the samples, 75% (n=15) were contaminated with Campylobacter spp., 70% (n=14) were with Salmonella species and 85% (n=17) were contaminated with Escherichia coli. <> Development of multiplex real time PCR with internal amplification control for simultaneous detection of Salmonella and Cronobacter in powdered infant formula. Lu J., Li L., Fan F., et al. x3R235W(*T0PR0T(Y@@QC= P AJ 1RpW <> x3R235W(*T0PR0T(Y@@QC= P AJ 1VpW A Loop-mediated isothermal amplification-based method for analysing animal feed for the presence of Salmonella. Besides, Salmonella virulence plasmid plays a crucial role in enhancing the ability of particular serovars to multiply in tissues outside the intestinal tract [19]. 1 0 obj J. Appl. stream Diagnostic real-time PCR for detection of Salmonella in food. campylobacter . 23 0 obj A high prevalence of orgA, prgH, and spaN genes in poultry and human isolates were observed in this study (71100%). Results were statistically significant compared to Salmonella culture positivity. Intra- and inter-run reproducibility was high for Salmonella spp. Rapid and specific detection of Salmonella spp in animal feed samples by PCR after culture enrichment. x3R235W(*T0PR0T(Y@@QC= P AJ 1PpW Appl. endstream endobj endobj MacKenzie K. D., Palmer M. B., Kster W. L., White A. P. Examining the link between biofilm formation and the ability of pathogenic Salmonella strains to colonize multiple host species. 1. endobj stream <> Rahn K., De Grandis S. A., Clarke R. C., et al. The qPCR sensitivity was comparable to that obtained with several in house qPCR targeting the Salmonella spp. %PDF-1.5
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In summary, this study provided a better insight into the epidemiology and pathogenicity of Salmonella serovars circulating in two Colombia regions. For example, PCR-based methods can be employed for the detection of pathogenic E. coli and Salmonella in beef [ 28 ]. Prevalence and profile of Salmonella from samples along the production line in Chinese beef processing plants. <> doi: 10.1128/AEM.02112-05, Yang, B., Qiao, L., Zhang, X., Cui, Y., Xia, X., Cui, S., et al. 0000012297 00000 n
strains, isolated from beef in Greater Tunis (Tunisia). The remaining 364 food samples were negative for Salmonella by both methods showing a relative qPCR specificity of 76.6 %. Protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) are described. Volume 30, Issue 1, March 2013, Pages 76-85. Microbiol.
0000013441 00000 n
endobj stream endobj Prevalence, virulence genes and antimicrobial resistance profiles of Salmonella serovars from retail beef in Selangor, Malaysia. J. endstream invA region (Gonzlez-Escalona et al., 2009; Hyeon et al., 2010; Zheng et al., 2014). The IAC DNA was added into the PCR reaction mix during all qPCR assays and was detected as expected. The combined MIL-RPA approach enabled detection of Salmonella at levels as low as 10 3 CFU mL 1. J. A pre-enrichment step was not included in this experiment. 7, 844856. Webthe detection of Salmonella (Fach et al., 1999). Guiney D. G., Fang F. C., Krause M., Libby S. Plasmid-mediated virulence genes in non-typhoid Salmonella serovars. A total of 500 food samples which were collected from local markets were processed during the study period. Animal welfare and microbiological safety of poultry meat: impact of different at-farm animal welfare levels on at-slaughterhouse Campylobacter and Salmonella contamination. Methods 70, 245251. x3R235W(*T0PR0T(Y@@QC= P AJ 14RpW endobj 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. endstream in different food products. and transmitted securely. Int. On the other hand, Webber et al. The molecular mechanisms of pathogenicity used by Salmonella involve genes, grouped in regions called pathogenicity islands that provide new characteristics that allow it to undergo a successful infective cycle [11]. %
0000004522 00000 n
Patterns I, III, V, and VIII were observed in poultry isolates. x\qqBDz endstream hU[O0O#_$)-VV6&!Lk4g;Imb:\|$">T2?LHEJ11C`. PCR demonstrated the specificity of invA primers for detection of Salmonella as confirmed by biochemical and serological assay and revealed that PCR was a rapid and useful tool for detection in food and beverage samples. Hierarchical effector protein transport by the Salmonella typhimurium SPI-1 Type III secretion system. stream 35 0 obj Coburn, B., Grassl, G. A., and Finlay, B. TABLE 3. A combination of immunomagnetic separation and polymerase chain reaction (IMS-PCR) was used to detect Salmonella in food samples and the shortened isolated from chicken faeces in Mafikeng, South Africa. <> Appl. 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. x_7*SO@\.Nsl@SO?08b FkVw6/!xQ[qbAC7)G]ULG`Dzw9IpawAr* uLt=i_F*jMwpaawi*4Nr@#& +r The PCR products could be confirmed by, A microtitration plate, antibody-capture, enzyme-linked immunosorbent assay was developed for detection of Salmonella typhimurium. endstream
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Real time PCR method for detection of Salmonella spp in environmental samples. <> (Table 1). 161, 1622. Serotyping, antimicrobial susceptibility, pulse field gel electrophoresis analysis of Salmonella isolates from retail foods in Henan Province, China. Some virulence genes may contribute to survival within the macrophage or intracellular survival, for example, pagC, spiA, msgA, and tolC genes [59]. Accessibility Though the prevalence of Salmonella in food has been mentioned previously in Tunisia, all studies have focused solely on positive culture isolates (Fendri et al., 2013; Oueslati et al., 2016). Cell. In 2018, the EU member states reported 5146 foodborne outbreaks where 33% correspond to illnesses caused by Salmonella [7]. Salmonella enterica Capture ELISA: Analysis of Food Samples A total of 84 samples belonging to different categories, i.e., dairy product, meat, pasta and flour, egg, and animal feed (Table 2) were analyzed by both the official method ISO 6579: 2002 and S. enterica capture ELISA. z(=a? ? Y_Q_Y__QGEt. Gel image of the PCR amplification of a DNA fragment from the invA gene (284bp) of representative Salmonella strains isolated from broiler farms. In this study, S. Anatum is the serovar most commonly found in raw red meat. The detection kit provides all the reagents required for the <> The method is time consuming (5 to 7 0000012210 00000 n
and . The participants were assigned to analyze the samples for detection of Campylobacter, Salmonella and Yersinia using the detection and characterization methods and Indicates significant differences between Salmonella positivity by culture compared to qPCR (P 0.05). 75, 37143720. 41 0 obj (2011). The limit of detection is 1 10 cfu / 25 g sample. 1Poultry Research Group, Laboratory of Immunology and Molecular Biology, Faculty of Veterinary Medicine and Zootechnics, University of Tolima, Santa Helena Highs, Postal Code 730006299, Ibagu, Tolima, Colombia, 2Immunobiology and Pathogenesis Research Group, Laboratory of Immunology and Molecular Biology, Faculty of Veterinary Medicine and Zootechnics, University of Tolima, Santa Helena Highs, A. <> 83:e00644-17. Food Control 38, 5460. Therefore, the ability to control bacterial pathogens in foods is very relevant, in particular the early detection of Salmonella contamination. QPCR results indicate that Salmonella contamination is common in milk and in different types of commercialized food. A. The qPCR assay exhibited a good intra and inter-run reproducibility. 42 0 obj x3R235W(*T0PR0T(Y@@QC= P AJ 14QpW In this study, 49 Salmonella enterica strains from the Bacterial Strain Collection of the Laboratory of Immunology and Molecular Biology were included, and Salmonella enteritidis (ATCC 13076) were used as a positive control. (2007). =&SX)e%'K9Dmk6kUFjae L and Listeria monocytogenes detection in food and environmental Lejeune P. Escherichia coli and Salmonella typhimurium: cellular and molecular biology. Wallis T. S., Galyov E. E. Molecular basis of salmonella-induced enteritis. 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 <>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/Annots[ 19 0 R 27 0 R 29 0 R 30 0 R 33 0 R 35 0 R 36 0 R 37 0 R 40 0 R 41 0 R 42 0 R 43 0 R 44 0 R 47 0 R 48 0 R 49 0 R 50 0 R 51 0 R 52 0 R 53 0 R 54 0 R 55 0 R 56 0 R 57 0 R 58 0 R 59 0 R 60 0 R 62 0 R 63 0 R] /MediaBox[ 0 0 612 792] /Contents 4 0 R/Group<>/Tabs/S/StructParents 0>>
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endobj McGhie E. J., Brawn L. C., Hume P. J., Humphreys D., Koronakis V. Salmonella takes control: effector-driven manipulation of the host. 21 0 obj Methods 4, 259267. Regarding the IAC, 320 nM IAC primers, 160 nM IAC probe, and 0.5 l of IAC DNA template (0.5 pg l-1, approximately 102 to 103 copies per PCR reaction) were added per reaction as described previously (Deer et al., 2010). E. coli isolates appear as blue and violet on ECC (Figure 2). Food Microbiol. <> Follow the instructions below to avoid nuclease-, *v8@|YiS9hjZ>KF;%)n,/ja-z5Mv[ ,- D]@^gbW:4/
}l#^jK> L=j H+Nc>20b$<6yt Fd C'lEvO0va15/FV[A!\9 -/.", |UaIj7fV:^hR]mTFw1?7 d9$ 1!gS$r~? V
e6I3&O#5};*d7EgHo.Iik:#'Wn5Y(AI*023. S. kentucky, S. Anatum, S. Altona, and S. Manchester were the most frequent serotypes, accounting for 28, 20, 12, and 12%, respectively, of all isolates. Ruiz M. J., Ramallo G., Colello R., et al. (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. Al-Khafaji N. S., Al-Bayati A. M., Al-Dahmoshi H. O. Virulence factors of Salmonella typhi in Salmonella spp. Steenackers H., Hermans K., Vanderleyden J., De Keersmaecker S. C. J. Salmonella biofilms: an overview on occurrence, structure, regulation and eradication. endobj The sipB, invA, orgA, prgH, and spaN genes are associated to the structure of TTSS, which allows Salmonella to invade phagocytic and nonphagocytic cells [40, 43]. stream WebThe results support the adequacy of the ISO for the detection of Salmonella spp. Patterns of virulence genes of Salmonella isolates obtained from poultry farms and cases of gastroenteritis in humans. Lipocalin-2 resistance confers an advantage to Salmonella enterica serotype typhimurium for growth and survival in the inflamed intestine. Indeed, a positive result at the lowest tested contamination level of Salmonella spp. On the other hand, food contamination with Salmonella may be underestimated using culture technique when food contained natural compounds with antimicrobial properties that could interfere with Salmonella detection (Jean-Gilles Beaubrun et al., 2016). In the present study, all the isolates carried the lpfA and csgA genes. The developed TaqMan qPCR assay including newly designed primer and probe sets for Salmonella spp. Genes related to survival inside cells functions such as pagC, spiA, msgA, and tolC were present in 97.4% (n=38/39), 97.4% (n=38/39), 97.4% (n=38/39), and 92.3% (n=36/39) of the poultry strains that were analyzed. sMr5r`l3jOO5sLW9'
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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. The Ct values of the experiment ranged from 18 0.28 to 35.27 0.70. Before applying our qPCR assay to food samples, a prior determination of the linear range and detection level was assessed using a known numbers of cloned PCR targets. Microbiol. /Contents 30 0 R>> Primers and probes provide specific detection of Salmonella-, S. Enteritidis-, S. Typhimurium-DNA in food, feed and environmental Received 2022 Nov 16; Revised 2023 Jan 30; Accepted 2023 Feb 2. Salmonella species are ubiquitous pathogens that are considered the major agents of foodborne disease worldwide [27, 28]. 186, 613. The high frequency of the spiA gene in poultry and human samples is considered critical due to the function of the gene that is related to the ability of the Salmonella serotypes to produce biofilms [25]. endobj
For example, genes such as SopB/SigD and SopE2 allow a rapid internalization of the bacteria playing an important role in Salmonella virulence [16]. detection by conventional culture and qPCR methods showed a difference statistically significant (P 0.001) (Table 3). Jones R. M., Wu H., Wentworth C., Luo L., Collier-Hyams L., Neish A. S. Salmonella AvrA coordinates suppression of host immune and apoptotic defenses via JNK pathway blockade. on two chromogenic media: Salmonella enteritidis is a colorless colony on ECC(A), Salmonella typhi & Salmonella typhimurium are pink Prevalence of different Salmonella serovars isolated from different types of food samples. According to this, the study aims to characterize the virulence factors of Salmonella spp. 74, 12991304. With the addition of PMA, the DNA 43 0 obj Lfstrm, C., Knutsson, R., Axelsson, C. E., and Radstrom, P., (2004). A real-time PCR for the detection of Salmonella enteritidis in poultry meat and consumption eggs. stream stream J. /Length 6481 >> startxref
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x3R235W(*T0PR0T(Y@@QC= P AJ 1TpW Enterobacteriaceace in Poultry Disease, 3rd Edn. WebDetection and quantification of Salmonella species in food samples is routinely performed using conventional culture-based techniques, which are labor intensive, involve well 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. <>/Group <> Probes 17, 215221. (B) Inter and intra-run reproducibility assessed using 2.00 100 to 2.00 105 copies per reaction in three independent runs. <> 13 0 obj InvA DNA was detected in 25 of 25 Salmonella positive specimens (100% sensitivity) and in 111 of 475 Salmonella culture negative food samples (23.4%) by qPCR (Table 3). Iannetti L., Neri D., Santarelli G. A., et al. Detection of Salmonella and Cronobacter in powdered infant formula virulence factors of Salmonella contamination Salmonella are shown Table... E. 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