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Development of Defined Microbial Population Standards Using Fluorescence Activated Cell Sorting for the Absolute Quantification of S. aureus Using Real-Time PCR

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Abstract

In this article, four types of standards were assessed in a SYBR Green-based real-time PCR procedure for the quantification of Staphylococcus aureus (S. aureus) in DNA samples. The standards were purified S. aureus genomic DNA (type A), circular plasmid DNA containing a thermonuclease (nuc) gene fragment (type B), DNA extracted from defined populations of S. aureus cells generated by Fluorescence Activated Cell Sorting (FACS) technology with (type C) or without purification of DNA by boiling (type D). The optimal efficiency of 2.016 was obtained on Roche LightCycler® 4.1. software for type C standards, whereas the lowest efficiency (1.682) corresponded to type D standards. Type C standards appeared to be more suitable for quantitative real-time PCR because of the use of defined populations for construction of standard curves. Overall, Fieller Confidence Interval algorithm may be improved for replicates having a low standard deviation in Cycle Threshold values such as found for type B and C standards. Stabilities of diluted PCR standards stored at −20°C were compared after 0, 7, 14 and 30 days and were lower for type A or C standards compared with type B standards. However, FACS generated standards may be useful for bacterial quantification in real-time PCR assays once optimal storage and temperature conditions are defined.

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Acknowledgments

This study was financially supported by the Department of Agriculture, Fisheries and Food under Food Institutional Research Measure (FIRM), NDP 2000-2006.

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  1. Ultan P. Cronin

    Present address: Flow Cytometry Unit Spanish National Cancer Research Centre (CNIO), c/ Melchor Fernández Almagro, 3, 28029, Madrid, Spain

Authors and Affiliations

  1. Department of Life Sciences, University of Limerick, Schrodinger Building, Castletroy, Co, Limerick, Ireland

    Alice Martinon, Ultan P. Cronin & Martin G. Wilkinson

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  1. Alice Martinon
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Correspondence to Martin G. Wilkinson.

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Martinon, A., Cronin, U.P. & Wilkinson, M.G. Development of Defined Microbial Population Standards Using Fluorescence Activated Cell Sorting for the Absolute Quantification of S. aureus Using Real-Time PCR. Mol Biotechnol 50, 62–71 (2012). https://doi.org/10.1007/s12033-011-9417-3

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