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Evaluation of concentration efficiency of the Pseudomonas aeruginosa phage PP7 in various water matrixes by different methods

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Abstract

Enteric viruses monitoring in surface waters requires the concentration of viruses before detection assays. The aim of this study was to evaluate different methods in terms of recovery efficiencies of bacteriophage PP7 of Pseudomonas aeruginosa, measured by real-time PCR, using it as a viral control process in water analysis. Different nucleic acid extraction methods (silica–guanidinium thiocyanate, a commercial kit (Qiagen Viral RNA Kit) and phenol–chloroform with alcohol precipitation) exhibited very low recovery efficiencies (0.08–4.18 %), being the most efficient the commercial kit used for subsequent experiments. To evaluate the efficiency of three concentration methods, PBS (as model for clean water) and water samples from rivers were seeded to reach high (HC, 106 pfu ml−1) and low concentrations (LC, 104 pfu ml−1) of PP7. Tangential ultrafiltration proved to be more efficient (50.36 ± 12.91, 17.21 ± 9.22 and 12.58 ± 2.35 % for HC in PBS and two river samples, respectively) than adsorption–elution with negatively charged membranes (1.00 ± 1.34, 2.79 ± 2.62 and 0.05 ± 0.08 % for HC in PBS and two river samples, respectively) and polyethylene glycol precipitation (15.95 ± 7.43, 4.01 ± 1.12 and 3.91 ± 0.54 %, for HC in PBS and two river samples, respectively), being 3.2–50.4 times more efficient than the others for PBS and 2.7–252 times for river samples. Efficiencies also depended on the initial virus concentration and aqueous matrixes composition. In consequence, the incorporation of an internal standard like PP7 along the process is useful as a control of the water concentration procedure, the nucleic acid extraction, the presence of inhibitors and the variability of the recovery among replicas, and for the calculation of the sample limit of detection. Thus, the use of a process control, as presented here, is crucial for the accurate quantification of viral contamination.

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Acknowledgments

This research was part of the project PICT-Red 276/06 funded by the Agencia Nacional de Promoción de Ciencia y Técnica in Argentina (ANPCyT). This project was partially supported by NIH Grant D43 TW005718 funded by the Fogarty International Center and the National Institute of Environmental Health Sciences, USA. Hugo Ramiro Poma, Patricia Angélica Barril and Gisela Masachessi received fellowships from CONICET and María Dolores Blanco Fernández from ANPCyT.

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Authors and Affiliations

  1. INIQUI-CONICET, Universidad Nacional de Salta, Av. Bolivia 5150, Salta, 4400, Argentina

    Hugo Ramiro Poma & Verónica Beatriz Rajal

  2. Fogarty International Center, University of California at Davis, One Shields Av., Davis, CA, 95616, USA

    Verónica Beatriz Rajal

  3. Cátedra de Virología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 4to piso, Ciudad Autónoma de Buenos Aires, 1113, Argentina

    María Dolores Blanco Fernández & Viviana Andrea Mbayed

  4. Instituto de Virología Dr. J. M. Vanella, Universidad Nacional de Córdoba, Córdoba, Argentina

    Patricia Angélica Barril, Miguel Oscar Giordano, Gisela Masachessi, Laura Cecilia Martínez, María Beatriz Isa & Silvia Viviana Nates

  5. Departamento Científico Pericial, Prefectura Naval Argentina, Benjamin Lavaisse 1058, Ciudad Autónoma de Buenos Aires, 1107, Argentina

    Gabriela López Riviello

  6. Instituto Nacional de Enfermedades Infecciosas INEI-ANLIS Dr. Carlos G. Malbrán, Velez Sarsfield 563, Ciudad Autónoma de Buenos Aires, 1281, Argentina

    María Cecilia Freire & Daniel Cisterna

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  1. Hugo Ramiro Poma
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  2. Verónica Beatriz Rajal
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Correspondence to Verónica Beatriz Rajal.

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Hugo Ramiro Poma and Verónica Beatriz Rajal shared first authorship.

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Poma, H.R., Rajal, V.B., Blanco Fernández, M.D. et al. Evaluation of concentration efficiency of the Pseudomonas aeruginosa phage PP7 in various water matrixes by different methods. Environ Monit Assess 185, 2565–2576 (2013). https://doi.org/10.1007/s10661-012-2731-9

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