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Journal of Clinical Microbiology, November 2000, p. 4215-4218, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Detection of Legionella pneumophila Using a Real-Time PCR Hybridization Assay

A. L. Ballard,1 N. K. Fry,2 L. Chan,3 S. B. Surman,3 J. V. Lee,3 T. G. Harrison,2 and K. J. Towner1,*

Department of Microbiology and PHLS Laboratory, University Hospital,1 and PHLS Water and Environmental Microbiology Research Unit,3 Queen's Medical Centre, Nottingham NG7 2UH, and Respiratory and Systemic Infection Laboratory, PHLS Central Public Health Laboratory, London NW9 5HT,2 United Kingdom

Received 29 March 2000/Returned for modification 8 June 2000/Accepted 18 August 2000


    ABSTRACT
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A real-time PCR hybridization assay for Legionella pneumophila is described; the assay uses LightCycler (Idaho Technology) methodology to specifically detect 2.5 CFU/reaction, equivalent to 1,000 CFU/liter of starting water sample. The assay, including DNA extraction and confirmation of product identity, is completed within 90 min of receipt of a sample.


    TEXT
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Legionnaires' disease is normally acquired by inhalation or aspiration of Legionella pneumophila serogroup 1 from a contaminated environmental source. Rapid identification of the source of infection is essential to prevent further cases of disease, and a number of conventional PCR assays for the detection of Legionella spp. have been described previously (6-10, 12-17). The introduction of rapid thermal cyclers combined with microvolume fluorimeters (e.g., the LightCycler; Idaho Technology, Idaho Falls, Idaho) now enables >30 PCR cycles in <20 min, combined with immediate confirmation of PCR product identity. This paper describes a prototype real-time assay using LightCycler methodology that detects L. pneumophila within 90 min of receipt of water samples.

Primers mip-Lpn0901F (5'-AACCGATGACACATCATTA) and mip-Lpn1011R (5'-CTTGCATGACTTTAGCCA) were designed to amplify a 131-bp region at the 5' end of the macrophage infectivity potentiator (mip) gene of L. pneumophila (3). These were used in conjunction with mip-specific hybridization probe mip-Lpn0941P (5'-Cy5-TCGGCACCAATGCTATAAGA-biotin).

Organisms used to assess the specificity of the assay are listed in Table 1. DNA was extracted from a single colony in 180 µl of lysis (ATL) buffer in a DNA Mini Kit (QIAgen Ltd., Crawley, United Kingdom) to a final eluate of 50 µl. One-liter natural water samples were concentrated (4) to a final volume of ca. 1 ml, of which 80 µl was added to 100 µl of ATL buffer and processed using the QIAgen kit to a final DNA eluate of 50 µl. DNA from laboratory-maintained water microcosms was extracted by centrifuging a 1-ml sample at 5,000 × g for 5 min, discarding 920 µl of supernatant, and, following resuspension of the pellet, processing the remaining 80 µl with the QIAgen kit as before. All DNA eluates were diluted 1:10 in 0.2% (wt/vol) bovine serum albumin (Sigma, Poole, United Kingdom) to minimize inhibition (11). For sensitivity testing and construction of a standard curve, samples from a 1-liter microcosm containing L. pneumophila serogroup 1 were prepared and the viable count was determined by plating out 100-µl portions of appropriate dilutions on buffered charcoal yeast extract agar plates (5).

                              
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  		TABLE 1.   Strains used for testing the specificity of the L. pneumophila LightCycler assay

Reaction mixtures contained 1 µl of DNA extract, 0.7 µl of LightCycler master mixture (Roche Diagnostics, Lewes, United Kingdom), 4 mM MgCl2, 3 pmol each of the mip primers, 3 pmol of hybridization probe, SYBR green (Biogene, Cambridge, United Kingdom) at a final concentration of 1:10,000, 0.1 U of uracil-N-glycosylase (Roche Diagnostics), and PCR grade water to a final volume of 7 µl. Of this, 5 µl was added by brief centrifugation to a LightCycler capillary reaction cuvette and amplified in a model LC32 Idaho Technology LightCycler. Reaction conditions were 3 min at 95°C, followed by 50 cycles of 0 s (hold time on reaching temperature) at 95°C, 1 s at 60°C, and 2 s at 72°C. The double-stranded PCR product was measured during the 60°C annealing step by detection of fluorescence associated with the binding of SYBR green dye to the product. Product identity was confirmed by fluorescence resonance energy transfer from SYBR green to the Cy5-labeled hybridization probe (1). The product melt was as follows: 0 s (hold time on reaching temperature) at 95°C, 0 s at 50°C, and 0 s at 95°C. Temperature change rates were 20°C/s, except for the final step, which had a temperature change rate of 0.2°C/s. Reactions were monitored on-line in real time.

The L. pneumophila-specific PCR product had a characteristic melting curve (melting temperature [Tm], 80°C) as monitored by SYBR green dissociation, with a specific melt (Tm, 63°C) of the Cy5-labeled hybridization probe (Fig. 1). Only strains of L. pneumophila generated specific PCR product. No PCR product was produced with other Legionella spp. or any of the other organisms tested (Table 1). Figure 2 shows the results obtained with different dilutions of the known culture of L. pneumophila. Quantification was achieved at a lower limit of 2.5 CFU/LightCycler reaction, equivalent to ca. 1,000 CFU/liter in the original water sample. In contrast, the sensitivity limit of a conventional gel-based multiplex PCR assay using the mip primers and a set of genus-specific 5S ribosomal DNA primers (2) was 25 CFU/reaction (i.e., an order of magnitude less sensitive than the LightCycler assay).


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  		FIG. 1.   Melting curves for L. pneumophila-specific LightCycler-based PCR. Shown are changes in SYBR green (A) and Cy5 (B) fluorescence (-dF/dT) versus temperature. ------, L. pneumophila serogroup 1; -----, negative control.


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  		FIG. 2.   Quantification of L. pneumophila-specific double-stranded PCR product as measured by SYBR green fluorescence. (A) Log fluorescence versus cycle number (a, 1,000 CFU/reaction; b, 100 CFU/reaction; c, 25 CFU/reaction; d, 2.5 CFU/reaction); (B) crossing points (cycle numbers) of log linear correlations with the noise line band, plotted against the logarithmic concentration of the standards.

The prototype LightCycler assay was further assessed with 14 natural water samples and 10 laboratory microcosms, of which 11 water samples and all 10 microcosms were culture positive for L. pneumophila. All 10 microcosms were also positive for L. pneumophila with the LightCycler assay. Six of the 11 culture-positive natural water samples were positive with the LightCycler assay, but of the 5 culture-positive samples that were negative, 3 contained <200 CFU/liter (i.e., below the detection limit of the LightCycler assay). These samples yielded positive assay results after being spiked with the L. pneumophila DNA extract (equivalent to 1,000 CFU) used to prepare the standard curve. The remaining two culture-positive samples appeared to contain PCR inhibitors, as they yielded a negative result even after being spiked. The three culture-negative water samples were also negative with the LightCycler assay but did not contain PCR inhibitors, as they yielded a positive result after being spiked.

In conclusion, the prototype LightCycler assay is a promising quantifiable biprobe method that amplifies a 131-bp region at the 5' end of the mip gene and appears to be specific for L. pneumophila. The sensitivity limit of 2.5 CFU/reaction (1,000 CFU/liter of starting water) probably represents ca. 25 copies of target DNA, since culture, following concentration by filtration and centrifugation, only detects about 10 to 30% of the bacteria present in the original sample, with the rest being either nonviable or lost during the concentration steps. No false-positive results compared with culture were obtained, but there were two false-negative results, apparently caused by PCR inhibition. The problem of inhibition, particularly that caused by iron compounds (e.g., rust) often present in environmental water samples, may limit the usefulness of PCR-based assays unless improved DNA preparation methods are developed and inhibition controls are routinely included. The sensitivity and specificity of the assay remain to be established with a larger number of varied samples, but the assay may be particularly useful in outbreak situations where a reservoir of infection normally contains >1,000 CFU/liter and there is a requirement to screen significant numbers of samples in as short a time as possible.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and PHLS Laboratory, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom. Phone: 44-115-9709163. Fax: 44-115-9422190. E-mail: Kevin.Towner{at}nottingham.ac.uk.


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Journal of Clinical Microbiology, November 2000, p. 4215-4218, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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