Development and clinical evaluation of an internally controlled, single-tube multiplex real-time PCR assay for detection of Legionella pneumophila and other Legionella species

Abstract

A multiplex real-time PCR assay for detection of Legionella pneumophila and Legionella spp. and including an internal control was designed. Legionella species, L. pneumophila, and the internal control were detected simultaneously by probes labeled with 6-carboxy-fluorescein, hexachlorofluorescein, and indodicarbocyanine, respectively. Therefore, no postamplification analysis was required in order to distinguish the targets. The sensitivity of both assays was 2.5 CFU/ml, and from analysis of 10 culture-positive and 74 culture-negative samples from patients investigated for legionellosis, 100% agreement was observed by both assays in comparison to culture. Four additional positives were found by the multiplex real-time PCR assay in the Legionella culture-negative samples.

FIG. 1.

(A) Relative fluorescence units obtained for a 10-fold dilution series of L. pneumophila ATCC 33152 from 10⁷ to 10² CFU/ml for the Legionella spp. assay. Each dilution gives a Ct value which can be plotted against the CFU to obtain a standard curve. The value obtained for the slope of the line gives a value for PCR efficiency; in this case it is 100%. (B) Relative fluorescence units obtained for a 10-fold dilution series of L. pneumophila ATCC 33152 from 10⁷ to 10² CFU/ml for the L. pneumophila assay. Each dilution gives a Ct value which can be plotted against the CFU to obtain a standard curve. The value obtained for the slope of the line gives a value for PCR efficiency; in this case it is 102.7%.

FIG. 2.

Determination of results. Each panel displays the readout from the multiplex PCR with the fluorophore indicated. (A) Sample positive for L. pneumophila, showing fluorescence with FAM (Legionella spp.), HEX (L. pneumophila), and Cy-5 (internal control). (B) Sample positive for Legionella spp. but not L. pneumophila, showing fluorescence with FAM (Legionella spp.) and Cy-5 (internal control). (C) Negative samples, showing fluorescence only with Cy-5 (internal control).