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

  • Gili Aviv
  • Ohad Gal-MorEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1734)


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).

Key words

Real-time PCR Reverse transcription cDNA SYBR green dye ROX Transcription Regulation Gene expression 



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.


  1. 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 5:391CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Fabrega A, Vila J (2013) Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clin Microbiol Rev 26:308–341CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Clements M, Eriksson S, Tezcan-Merdol D et al (2001) Virulence gene regulation in Salmonella enterica. Ann Med 33:178–185CrossRefPubMedGoogle Scholar
  4. 4.
    Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193CrossRefPubMedGoogle Scholar
  5. 5.
    Ginzinger DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30:503–512CrossRefPubMedGoogle Scholar
  6. 6.
    Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. BioTechniques 39:75–85CrossRefPubMedGoogle Scholar
  7. 7.
    Navarro E, Serrano-Heras G, Castano MJ, Solera J (2015) Real-time PCR detection chemistry. Clin Chim Acta 439:231–250CrossRefPubMedGoogle Scholar
  8. 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 84:901–910CrossRefPubMedGoogle Scholar
  9. 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:e01395-16CrossRefPubMedPubMedCentralGoogle Scholar
  10. 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 16:977–994CrossRefPubMedGoogle Scholar
  11. 11.

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.The Infectious Diseases Research LaboratorySheba Medical CenterTel-Hashomer, Ramat GanIsrael
  2. 2.Department of Clinical Microbiology and Immunology, Sackler Faculty of MedicineTel Aviv UniversityTel AvivIsrael

Personalised recommendations