Development of defined microbial population standards using fluorescence activated cell sorting for the absolute quantification of S. aureus using real-time PCR.

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.

journal_name

Mol Biotechnol

journal_title

Molecular biotechnology

authors

Martinon A,Cronin UP,Wilkinson MG

doi

10.1007/s12033-011-9417-3

subject

Has Abstract

pub_date

2012-01-01 00:00:00

pages

62-71

issue

1

eissn

1073-6085

issn

1559-0305

journal_volume

50

pub_type

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