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

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-μl 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 μm-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 its DNA is a common contaminant of necessary reagents.

Introduction

Real-time PCR, a rapid and quantitative method, is increasingly popular in microbial pathogen detection from environmental and clinical samples. While the detection limit can be as low as one genome, DNA contamination presents a potential obstacle to achieving this low detection limit. For example, contamination in genomic DNA preparations causing false positives using conventional PCR methods has confounded clinical bacterial detection as well as culture-independent microbial surveys of environmental samples [1], [2]. In the application of real-time PCR, water and reagents contaminated with trace amounts of DNA have even greater potential for artifacts, due to the greater sensitivity of real-time PCR detection.

Several groups have described real-time PCR assays for the detection of L. pneumophila [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15] and some reported detection limits to be as low as 2.5 CFU or 3–10 genomes per reaction [3], [6], [9] all using fluorescently labeled hybridization probes in conjunction with the LightCycler system (ABI).

Our study initially attempted to develop a real-time PCR assay for the detection of L. pneumophila from evaporative cooler waters in Socorro, New Mexico, in the southwestern US, where evaporative coolers are used for air conditioning. Using primers targeting the L. pneumophila-specific mip gene [3], and SYBR Green detection in conjunction with the iCycler iQ real-time PCR detection system (Bio-Rad, Hercules, CA), we could detect as little as 4.8 fg L. pneumophila genomic DNA, equivalent to 1.3 copies of the mip gene, from serially diluted, purified genomic DNA in a total reaction volume of 50 μl. However, the negative controls, i.e. no-template, also yielded cycle threshold (C_T) in real-time PCR and DNA product of the appropriate size observed in gel electrophoresis. This low-level contamination persisted when individual reagents in real-time PCR were replaced. 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. Based on results from exhaustive negative controls, we have traced the contamination to a commercial reagent (water) used in real-time PCR.