Advertisement

Using RT qPCR for Quantifying Mycobacteria marinum from In Vitro and In Vivo Samples

  • Han Xaio
  • Stephen H. Gillespie
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1736)

Abstract

Mycobacterium marinum, the causative agent of fish tuberculosis, is rarely a human pathogen causing a chronic skin infection. It is now wildely used as a model system in animal models, especially in zebra fish model, to study the pathology of tuberculosis and as a means of screening new anti-tuberculosis agent. To facilitate such research, quantifying the viable count of M. marinum bacteria is a crucial step. The main approach used currently is still by counting the number of colony forming units (cfu), a method that has been in place for almost 100 years. Though this method well established, understood and relatively easy to perform, it is time-consuming and labor-intensive. The result can be compromised by failure to grow effectively and the relationship between count and actual numbers is confused by clumping of the bacteria where a single colony is made from multiple organisms. More importantly, this method is not able to detect live but not cultivable bacteria, and there is increasing evidence that mycobacteria readily enter a “dormant” state which confounds the relationship between bacterial number in the host and the number detected in a cfu assay. DNA based PCR methods detect both living and dead organisms but here we describe a method, which utilizes species specific Taq-Man assay and RT-qPCR technology for quantifying the viable M. marinum bacterial load by detecting 16S ribosomal RNA (16S rRNA).

Key words

Treatment monitoring Antibiotic resistance Mycobacterium marinum Molecular diagnostics 

Notes

Acknowledgments

The research leading to this chapter was supported by the Innovative Medicines Initiative Joint Undertaking under grant agreement no 115337, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in-kind contribution.

References

  1. 1.
    Honeyborne I, McHugh TD, Phillips PPJ et al (2011) Molecular bacterial load assay, a culture-free biomarker for rapid and accurate quantification of sputum mycobacterium tuberculosis bacillary load during treatment. J Clin Microbiol 49:3905–3911CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Honeyborne I, Mtafya B, Phillips PPJ et al (2014) The molecular bacterial load assay replaces solid culture for measuring early bactericidal response to antituberculosis treatment. J Clin Microbiol 52:3064–3067CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Billington OJ, McHugh TD, Gillespie SH (1999) Physiological cost of rifampin resistance induced in vitro in Mycobacterium tuberculosis. Antimicrob Agents Chemother 43:1866PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.School of MedicineUniversity of St AndrewsSt AndrewsUK

Personalised recommendations