Detection Method for Salmonella Typhimurium and Salmonella Enteritidis using Real-Time Polymerase Chain Reaction

  • Abstract
  • Keywords
  • References
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  • Abstract

    Salmonella is a pathogenic bacterium that can cause serious harm to humans. Chicken carcasses have been reported contaminated by Salmonella, especially S. Typhimurium and S. Enteritidis. These two serovars are very difficult to be confirmed and distinguished using biochemical analysis, therefore a rapid method for detection and differentiation of both is required. The objective of this study was to evaluate designed primer for detection and differentiation of S. Typhimurium and S. Enteritidis on chicken carcasses using real time Polymerase Chain Reaction (rt-PCR). Detection of Salmonella spp. was conducted using primer sequence from invA gene. Differentiation of both Salmonella serovars was conducted using specific target gene from S. Typhimurium (STM) and specific virulence plasmid of S. Enteritidis (Prot6E). The result showed that invA primer effective to detect all species Salmonella tested and has good specificity that could not detect Escherichia coli and Shigella dysenteriae in the similar melting temperature.   Two specific primers STM and prot6E have distinguished between S. Typhimurium and S. Enteritidis.  Sensitivity of method showed very good with 0.5 μM primer concentration of invA, STM and prot6E that were 0.2 pg, 22 pg and 28 pg respectively. Initial trial showed that this method can be applied for detection of Salmonella spp. and two serovars in chicken carcasses.



  • Keywords

    chicken carcasses, detection method, real-time PCR, Salmonella Enteritidis, Salmonella Typhimurium

  • References

      [1] Brenner FW, Villar RG, Angulo FJ, Tauxe R, Swaminathan B, Salmonella nomenclature, J Clin Microbiol. (2003) 8:2465–2467

      [2] [FSAI] Food Safety Authority of Ireland, (2007), Microbiological quality of ice for cooling drinks, 1st National Microbiological Survey. Final Report.

      [3] Cummings PL, Sorvillo F, Kuo T, (2012), The Burden of Salmonellosis in the United States. In: Mahmoud BSM, editor. Salmonella A Dangerous Foodborne Pathogen. Shanghai (CN): InTech China. pp 1-20.

      [4] Malorny B, Made D, Charlotta L, (2013), Real-time PCR Detection of Food-borne Pathogenic Salmonella spp. In: Rodriguez DL, editor. Real-time PCR in Food Science. Norfolk (UK): Caister Academic Press. pp 57-78.

      [5] Silva DM and Domingues L, On the track for an efficient detection of Escherichia coli in water: a review on PCR based methods, Ecotoxicology and Environmental Safety. (2015), 113, 400-411.

      [6] Nurjanah S, Rahayu WP, Komalasari E, Sensitivity of multiplex real-time PCR assay for detection of pathogenic E. coli on ice sample. (2017), November 14-17; Ho Chi Minh City, Vietnam. Ho Chi Minh City (VI): Proceedings of the 15th ASEAN Conference on Food Science and Technology.

      [7] Gaywee J, Suzana R, James AH, Abdu FA, Transcriptional analysis of Rickettsia prowazekii invasion gene homolog (InVA) during host cell infection, Journal of Infection and Immmunity. 200270(11) : 6346-6354.

      [8] Shanmugasamy M, Velayutham T, Rajeswar J. InvA gene specific PCR for detection of Salmonella from broilers. Journal of Veteriner. (2014),(12): 562-564.

      [9] Rahn K, Grandis AD, Clarke RC, McEwen SA, Galan JE, Ginocchio C, Curtiss R, Gyles CL. Amplification of an invA gene sequence of Salmonella Typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Molecular and Cellular Probes. (1992), 6: 271-279.

      [10] Malorny B, Hoorfar J, Hugas M, Heuvelink A, Fach P, Ellerbroek L, Bunge C, Dorn C, Helmuth R, Inter-laboratory diagnostic accuracy of a Salmonella specific PCR-based method. Int. J. Food Microbiol. (2003), 89: 241-249.

      [11] Clavijo RI, Cindy L, Gary LA, Lee WR, Sangwei LU. Identification of genes associated with survival of Salmonella enterica serovar Enteritidis in chicken egg albumin. J. of App Envi Microbiol. (2006), 2(2): 1055-1064.

      [12] Bäumler AJ, Tsolis RM, Heffron F. Contribution of fimbrial operons to attachment to and invasion of epithelial cell lines by Salmonella typhimurium. Infect. Immun. (1996),64: 1862-1865.

      [13] Bakshi CS, Singh VP, Malik M, Singh RK, Sharma B, 60 kb plasmid and virulenceassociated genes are positively correlated with Salmonella Enteritidis pathogenicity in mice and chickens. Vet. Res. Commun.( 2003), 27: 425-432.

      [14] Reyes EL, Balam CM, Rodriguez BI, Valdes J, Kameyama L, Martinez PF. 2010. Purification of bacterial genomic DNA in less than 20 min using chelex-100 microwave: example from strains of lactic acid bacteria isolated from soil samples. Antonie van Leewenhoek. 98: 456-474.

      [15] Fukushima M, Kakinuma K, Kawaguchi R. 2002. Phylogenetic analysis of Salmonella, Shigella, and Escherichia coli strains on the basis of the gryB gene sequence. J. of Clin Microbiol. 40(8): 2779-2785

      [16] Hoorfar J, Ahrens P, Radstrom P. Automated 5’ nuclease PCR assay for identification of Salmonella enterica, J Clin Microbiol. (2000), 38(9):3429-3435.

      [17] Park SH, Kim HJ, Cho WH, Kim JH, Oh MH, Kim SH, Lee BK, Ricke SC, Kim HY, Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Tyohi using multiplex PCR, FEMS Microbiology Letters. (2009),301: 137-146.

      [18] Hadjinicolaou A, Dmetriou VL, Emmanuel MA, Kakoyiannis CK, Kostrikis LG, Molecular beacon-based real-time PCR detection of primary isolates of Salmonella Typhimurium and Salmonella Enteritidis in environmental and clinical samples, BioMedCentral Microbiology, (2009), 9(97): 1-14.

      [19] Pestana E, Belak S, Diallo A, Crowther JR, Viljoen GJ. 2010. Early, Rapid and Sensitive Veterinary Molecular Diagnostic Real-Time PCR Application. Wageningen (NL): Springer Netherland.

      [20] Singh P, Mustapha A, Development of a real-time PCR melt curve assay for simultaneous detection of virulent and antibiotic resistant Salmonella. Food Microbiology. (2014) 44: 6-14

      [21] Oliviera ACS, Rosa MC, Borchardt JL, Menegon YA, Fernandes MMA, Validating efficiency of a simplex PCR and Quantitative SYBR Green Qpcr FOR THE Identification of Salmonella spp. DNA, Journal of Food: Microbiology, Safety and Hygiene. (2018) 3(1): 1-4.

      [22] Gunnel MK, Lovelace CD, Satterfield BA, Moore EA, Kim LO, Robison RA, A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies, J Med Microbiol, (2012), 61: 1525-1531. DOI: 10.1099/jmm.0.046631-0.

      [23] Besson G & Kazanji M, One-step, multiplex, real-time PCR assay with molecular beacon probe for simultaneous detection, differentiation, and quantification of human T-cell leukimia virus type 1,2, and 3. Journal of Clinical Microbiology. 2009, 47 (4), 1129-1135.




Article ID: 27661
DOI: 10.14419/ijet.v7i4.14.27661

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