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Using RT qPCR for quantifying Mycobacteria marinum from in vitro and in vivo samples

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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).
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Details

Original languageEnglish
Title of host publicationAntibiotic Resistance Protocols
EditorsStephen Gillespie
Place of PublicationNew York, NY
PublisherHumana Press/Springer
Pages137-145
Number of pages9
ISBN (Electronic)9781493976386
ISBN (Print)9781493976362
DOIs
StatePublished - 2018

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume1736
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

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