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Real-Time Reverse Transcription PCR as a Tool to Study Virulence Gene Regulation in Bacterial Pathogens

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Host-Pathogen Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1734))

Abstract

Quantitative real-time PCR (qRT-PCR) is a highly sensitive and reliable method for detection and quantification of DNA. When combined with a prior stage of RNA reverse transcription to generate complementary DNA (cDNA), this is a powerful approach to determine and analyze gene transcriptional expression. Real-time quantitative reverse transcription PCR has become the gold standard method in studying genes expression and virulence regulation under various genetic backgrounds (e.g., in the absence of regulators) or environmental conditions. Here we demonstrate the utilization of this approach to study the transcriptional regulation of the conjugation pilus of the Salmonella enterica serovar Infantis virulence plasmid (pESI).

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References

  1. Gal-Mor O, Boyle EC, Grassl GA (2014) Same species, different diseases: how and why typhoidal and non-typhoidal salmonella enterica serovars differ. Front Microbiol 5:391

    Article  PubMed  PubMed Central  Google Scholar 

  2. Fabrega A, Vila J (2013) Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clin Microbiol Rev 26:308–341

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Clements M, Eriksson S, Tezcan-Merdol D et al (2001) Virulence gene regulation in Salmonella enterica. Ann Med 33:178–185

    Article  CAS  PubMed  Google Scholar 

  4. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193

    Article  CAS  PubMed  Google Scholar 

  5. Ginzinger DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30:503–512

    Article  CAS  PubMed  Google Scholar 

  6. Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. BioTechniques 39:75–85

    Article  CAS  PubMed  Google Scholar 

  7. Navarro E, Serrano-Heras G, Castano MJ, Solera J (2015) Real-time PCR detection chemistry. Clin Chim Acta 439:231–250

    Article  CAS  PubMed  Google Scholar 

  8. Schefe JH, Lehmann KE, Buschmann IR et al (2006) Quantitative real-time RT-PCR data analysis: current concepts and the novel "gene expression's CT difference" formula. J Mol Med 84:901–910

    Article  CAS  PubMed  Google Scholar 

  9. Aviv G, Rahav G, Gal-Mor O (2016) Horizontal transfer of the Salmonella enterica serovar Infantis resistance and virulence plasmid pESI to the gut microbiota of warm-blooded hosts. MBio 7:e01395-16

    Article  PubMed  PubMed Central  Google Scholar 

  10. Aviv G, Tsyba K, Steck N et al (2014) A unique megaplasmid contributes to stress tolerance and pathogenicity of an emergent Salmonella enterica serovar Infantis strain. Environ Microbiol 16:977–994

    Article  CAS  PubMed  Google Scholar 

  11. QIAGEN (2012) RNeasy® Mini Handbook. https://www.qiagen.com/us/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbc-bf9f6fa33e24$lang=en

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Acknowledgments

The research in Gal-Mor lab is supported by a grant number 1096.39.11/2010 from the German-Israeli Foundation for Scientific Research and Development (GIF); by a grant number 999/14 from the Israel Science Foundation (ISF) and by grant number 3-0000-12435 from Infect-ERA and the Chief Scientist’s Bureau in the Israeli Ministry of Health.

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Correspondence to Ohad Gal-Mor .

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Aviv, G., Gal-Mor, O. (2018). Real-Time Reverse Transcription PCR as a Tool to Study Virulence Gene Regulation in Bacterial Pathogens. In: Medina, C., López-Baena, F. (eds) Host-Pathogen Interactions. Methods in Molecular Biology, vol 1734. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7604-1_3

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  • DOI: https://doi.org/10.1007/978-1-4939-7604-1_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7603-4

  • Online ISBN: 978-1-4939-7604-1

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