Chapter 49 : Rapid Quantification of by PCR

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To estimate the risk of legionellosis from the plumbing systems, it is a common practice to count CFU in water samples. Interestingly, can survive outside of protozoa for a longer period of time and can fall-still alive-into a viable but nonculturable (VBNC) physiological state, from which it can be recovered by addition of amoebae. The technique of quantitative competitive-template (QC)-PCR is mainly used in virology. The principle of QC-PCR is based on the simultaneous amplification of the genomic template and a recombined internal PCR standard (IS), which usually can be distinguished as the smaller template. There are a large number of useful PCR targets and primers for specific detection of . To demonstrate legionellae-specific QC-PCR in principle, the authors chose a 16S rRNA gene-based assay, which has proven to be reliable in the routine diagnostics of clinical microbiology and has recently been proposed as one method for identification of the most common species. QC-PCR could be evaluated as a fast and simple method for enumeration in infection models or in environmental water samples. This approach might even detect genomes, which cannot be recovered on agar plates because of low plating efficiency or organisms present in a VBNC state.

Citation: Jonas D, Sahlmüller G. 2002. Rapid Quantification of by PCR, p 257-259. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch49
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Image of FIGURE 1

Agarose gel-electrophoresis of quantitative-competitive PCR amplification products stained with ethidium bromide and visualized under UV illumination. The twofold dilution series of the competitive internal standard plasmid DNA ranged from 25 fg (lane 1) to 800 fg (lane 6). M, molecular size standard. Numbers of CFU are indicated above the gel.

Citation: Jonas D, Sahlmüller G. 2002. Rapid Quantification of by PCR, p 257-259. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch49
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