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Multiplex-PCR Assay for Detection of some Major Virulence Genes of Salmonella enterica Serovars from Diverse Sources


Affiliations
1 State Biotechnology Hub, Assam, College of Veterinary Science, Guwahati 781 022, India
2 Department of Biotechnology, Gauhati University, Guwahati 781 014, India
3 Department of Animal Biotechnology, College of Veterinary Science, Khanapara 781 022, India
4 Department of Microbiology, College of Veterinary Science, Khanapara 781 022, India
 

The virulence of Salmonella depends on virulence factors encoded by specific genes. The present study describes the development of a simple and rapid multiplex PCR (m-PCR) assay for simultaneous detection of seven major virulence genes of Salmonella (invA, invH, stn, sopB, sopE, sefC and pefA). Presence of these genes was studied using 17 standard cultures and 76 field isolates from different sources. Seventeen non-Salmonella strains were also tested for specificity of the optimized PCR. A spiked control experiment was done to detect the sensitivity of m-PCR assay. The primer pairs were found to be specific for Salmonella only. The assay detected 250 pg of purified chromosomal DNA, or 12 cfu of Salmonella in crude lysates. All the field isolates and standard strains of Salmonella were found to carry invA, invH, stn and sopB genes, while variability was observed with respect to sopE, sef C and pefA genes. Thus this multiplex PCR assay provides a simple and rapid method for detection of major virulence factors in important clinical serovars of Salmonella.

Keywords

Multiplex PCR, Salmonella, Spiking, Virulence Genes.
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  • Toboldt, A., Tietze, E., Helmuth, R., Junker, E., Fruth, A. and Malornya, B., Population structure of Salmonella enterica serovar 4, [5], 12: b: strains and likely sources of human infection. Appl. Environ. Microbiol., 2013, 79, 5121–5129.

  • Hensel, A., Evolution of pathogenicity islands of Salmonella enterica. Indian J. Med. Microbiol., 2004, 294, 95–102.

  • Wisner, A., Desin, T., White, A., Potter, A. and Köster, W., The Salmonella pathogenicity island-1 and-2 encoded type III secretion systems. In Salmonella – A Diversified Superbug (ed. Kumar, Y.), InTech, Croatia, 2012, pp. 469–494.

  • Sabbagh, S. C., Forest, C. G., Lepage, C., Leclerc, J. M. and Daigle, F., So similar, yet so different: uncovering distinctive features in the genomes of Salmonella enterica serovars Typhimurium and Typhi. FEMS Microbiol. Lett., 2010, 305, 1–13.

  • Galan, J. E., Ginocchio, C. and Costeas, P., Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. J. Bacteriol., 1998, 174, 4338–4349.

  • Clouthier, S. C., Collinson, S. K. and Kay, W. W., Unique fimbriaelike structures encoded by sef D of the SEF 14 fimbrial gene cluster of Salmonella Enteritidis. Mol. Microbiol., 1994, 12, 893– 901.

  • Chopra, A. K. et al., Role of Salmonella enterotoxin in overall virulence of the organism. Microb. Pathogenesis, 1999, 27, 155– 171.

  • Wood, M. W., Jones, M. A., Watson, P. R., Hedges, S., Wallis, T. S. and Galyov, E. E., Identification of a pathogenicity island required for Salmonella enteropathogenicity. Mol. Microbiol., 1998, 29, 883–891.

  • Wallis, T. S. and Galyov, E. E., Molecular basis of Salmonella induced enteritis. Mol. Microbiol., 2000, 36, 997–1005.

  • Ahmer, B., Tran, M. and Heffron, F., The virulence plasmid of Salmonella Typhimurium is self-transmissible. J. Bacteriol., 1999, 181, 1364–1368.

  • Rotger, R. and Casadesus, J., The virulence plasmids of Salmonella. Int. Microbiol., 1999, 2, 177–184.

  • Prager, R., Fruth, A. and Tschape, H., Salmonella enterotoxin (stn) gene is prevalent among strains of Salmonella enterica but not among Salmonella bongori and other Enterobacteriaceae. FEMS Immunol. Med. Microbiol., 1995, 12, 47–50.

  • Rahman, H., Prager, R. and Tschape, H., Occurrence of sef and pef genes among different serovars of Salmonella. Indian. J. Med. Res., 2000, 111, 40–42.

  • Alvarez, J., Sota, M., Vivanco, A. B., Perales, I., Cisterna, R., Rementeria, A. and Garaizar, J., Development of a multiplex PCR technique for detection and epidemiological typing of Salmonella in human clinical samples. J. Clin. Microbiol., 2004, 42, 1734– 1738.

  • de Freitas, C. G. et al., PCR multiplex for detection of Salmonella Enteritidis, Typhi and Typhimurium and occurrence in poultry meat. Int. J. Food Microbiol., 2010, 139, 15–22.

  • Saeki, E. K., Juliane, A. J., Bonfante, R. C., Elisa, Y., Hirooka, E. Y. and de Oliveira, T. C. R. M., Multiplex PCR (mPCR) for the detection of Salmonella spp. and the differentiation of the Typhimurium and Enteritidis serovars in chicken meat. J. Food Saf., 2013, 33, 25–29.

  • Theron, J., Morar, D., Preez, D. U., Brozel, M. V. S. and Venter, S. N., A sensitive seminested PCR method for the detection of Shigella in spiked environmental water samples. Water. Res., 2001, 35, 869–874.

  • Pathmanathan, S. G., Cardona-Castro, N., Sánchez-Jiménez, M. M., Correa-Ochoa, M. M., Puthucheary, S. D. and Thong, K. L., Simple and rapid detection of Salmonella strains by direct PCR amplification of the hilA gene. J. Med. Microbiol., 2003, 52, 773– 776.

  • Jamshidi, A., Bassami, M. R. and Afshari-Nic, S., Identification of Salmonella serovars Typhimurium by a multiplex PCR-based assay from poultry carcasses in Mashhad-Iran. Int. J. Vet. Res., 2008, 3, 43–48.

  • Malorny, B., Hoorfar, J., Bunge, C. and Helmuth, R., Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard. Appl. Environ. Microbiol., 2003, 69, 290.

  • Zou, W. et al., Microarray analysis of virulence gene profiles in Salmonella serovars from food/food animal environment. J. Infect. Dev. Ctries., 2011, 5, 94–105.

  • Rahman, H., Prevalence and phenotypic expression of sopB gene among clinical isolates of Salmonella enterica. Indian. J. Med. Res., 2006, 123, 83–88.

  • Rahman, H., Streckel, W., Prager, R. and Tschape, H., Prevalence of sopE gene & its phenotypic expression among different serovars of Salmonella isolated from man and animals. Indian. J. Med. Res., 2004, 120, 35–38.

  • Rahman, H., Hardth, W. D., Muruhkar, H. V. and Bhattacharyya, D. K., Occurrence of sopE gene and its phenotypic expression among different serovars of Salmonella enterica isolated from man and animals. Indian. J. Exp. Biol., 2005, 43, 631–634.

  • Suez, J. et al., Virulence gene profiling and pathogenicity characterization of non-typhoidal Salmonella accounted for invasive disease in humans. PLoS ONE, 2013, 8, e58449.

  • Murugkar, H. V., Rahman, H. and Dutta, P. K., Distribution of virulence genes in Salmonella serovars isolated from man and animals. Indian J. Med. Res., 2003, 117, 66–70.

  • Muthu, G. et al., Detection of virulence genes from Salmonella species in Chennai, India. CIB Tech. J. Microbiol., 2014, 3, 11– 14.

  • Lim, Y. H. et al., Multiplex polymerase chain reaction assay for selective detection of Salmonella enterica serovar Typhimurium. Jap. J. Infect. Dis., 2003, 56, 151–155.

  • Kapley, A., Lampel, K. and Purohit, H. J., Thermocycling steps and optimization of multiplex PCR. Biotechnol. Lett., 2000, 22, 1913–1918.

  • Khan, A. B., Sahir, K. H., Ahmed, M. and Khan, S. I., Rapid detection of Salmonella in food samples by polymerase chain reaction after a 10 h preenrichment. J. Food Safety, 2014, 34, 79–86.


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  • Multiplex-PCR Assay for Detection of some Major Virulence Genes of Salmonella enterica Serovars from Diverse Sources

Abstract Views: 455  |  PDF Views: 127

Authors

Mridusmita Choudhury
State Biotechnology Hub, Assam, College of Veterinary Science, Guwahati 781 022, India
Probodh Borah
State Biotechnology Hub, Assam, College of Veterinary Science, Guwahati 781 022, India
Hridip Kumar Sarma
Department of Biotechnology, Gauhati University, Guwahati 781 014, India
Luit Moni Barkalita
Department of Animal Biotechnology, College of Veterinary Science, Khanapara 781 022, India
Naba Kumar Deka
State Biotechnology Hub, Assam, College of Veterinary Science, Guwahati 781 022, India
Isfaqul Hussain
Department of Microbiology, College of Veterinary Science, Khanapara 781 022, India
Md. Iftikar Hussain
State Biotechnology Hub, Assam, College of Veterinary Science, Guwahati 781 022, India

Abstract


The virulence of Salmonella depends on virulence factors encoded by specific genes. The present study describes the development of a simple and rapid multiplex PCR (m-PCR) assay for simultaneous detection of seven major virulence genes of Salmonella (invA, invH, stn, sopB, sopE, sefC and pefA). Presence of these genes was studied using 17 standard cultures and 76 field isolates from different sources. Seventeen non-Salmonella strains were also tested for specificity of the optimized PCR. A spiked control experiment was done to detect the sensitivity of m-PCR assay. The primer pairs were found to be specific for Salmonella only. The assay detected 250 pg of purified chromosomal DNA, or 12 cfu of Salmonella in crude lysates. All the field isolates and standard strains of Salmonella were found to carry invA, invH, stn and sopB genes, while variability was observed with respect to sopE, sef C and pefA genes. Thus this multiplex PCR assay provides a simple and rapid method for detection of major virulence factors in important clinical serovars of Salmonella.

Keywords


Multiplex PCR, Salmonella, Spiking, Virulence Genes.

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DOI: https://doi.org/10.18520/cs%2Fv111%2Fi7%2F1252-1258