Sensitive, real-time PCR detects low-levels of contamination by Legionella pneumophila in commercial reagents

Molecular and Cellular Probes
Hua ShenThomas L Kieft

Abstract

In a real-time PCR assay of Legionella pneumophila (targeting the L. pneumophila-specific mip gene and using SYBR Green dye for DNA detection in conjunction with the iCycler system) we detected as few as 1.3 copies of a mip gene in a 50-microl reaction from serially diluted L. pneumophila genomic DNA. However, cycle threshold (C(T)) were yielded and DNA product detected in our no-template negative controls and the phenomenon persisted when two separate batches of PCR reagents and water from two different biochemical companies were tested. Since L. pneumophila can be widespread in municipal water supplies, the commercial reagents, especially the reagent water (80% of the reaction volume), could be the source of contamination. To test this hypothesis, we treated Millipore Milli-Q water by filtering through a 0.2 microm-pore-size polycarbonate filter to remove bacteria prior to autoclaving. Real-time PCR using this water had no contamination. Our finding is indirect evidence that commercially available purified water can harbor low level contamination by L. pneumophila DNA that has escaped purification processes. This presents a challenge when developing a sensitive DNA-based bacterial detection method if the target organism or it...Continue Reading

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Citations

Jun 19, 2008·Journal of Microbiological Methods·Paul G DennisPenny R Hirsch
Sep 25, 2007·Journal of Microbiological Methods·Maria B CadenasEdward B Breithschwerdt
Feb 19, 2008·Journal of Microbiological Methods·Sarah S SilkieKara L Nelson
Dec 19, 2013·BioTechniques·Tetsushi Suyama, Mamoru Kawaharasaki
Aug 24, 2016·Frontiers in Nutrition·Jacobo de la Cuesta-Zuluaga, Juan S Escobar
Mar 3, 2018·Genes·Raphael Eisenhofer, Laura S Weyrich
Apr 24, 2018·PLoS Computational Biology·Sean M GibbonsEric J Alm
Nov 20, 2019·International Journal of Cosmetic Science·R SfrisoR Campiche
May 10, 2017·Microbiome·Dorothy KimKyle Bittinger
Jan 17, 2020·The Journal of Pathology. Clinical Research·Caitlin A SelwayLaura S Weyrich
Feb 9, 2019·Scientific Reports·N WallyC Steininger
Dec 12, 2018·Frontiers in Microbiology·Olivia S HersheyHazel A Barton
Jul 7, 2021·Journal of Perinatal Medicine·Eunjung JungChaur-Dong Hsu

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