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

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• 作者：Hugo Ramiro Poma (1)
  Verónica Beatriz Rajal (1) (2)
  María Dolores Blanco Fernández (3)
  Patricia Angélica Barril (4)
  Miguel Oscar Giordano (4)
  Gisela Masachessi (4)
  Laura Cecilia Martínez (4)
  María Beatriz Isa (4)
  María Cecilia Freire (6)
  Gabriela López Riviello (5)
  Daniel Cisterna (6)
  Silvia Viviana Nates (4)
  Viviana Andrea Mbayed (3)
  
• 关键词：Virus concentration ; Surface water ; PP7 ; Absorption/elution ; Polyethylene glycol ; Ultrafiltration ; qRT ; PCR
• 刊名：Environmental Monitoring and Assessment
• 出版年：2013
• 出版时间：March 2013
• 年：2013
• 卷：185
• 期：3
• 页码：2565-2576
• 全文大小：212KB
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• 作者单位：Hugo Ramiro Poma (1)
  Verónica Beatriz Rajal (1) (2)
  María Dolores Blanco Fernández (3)
  Patricia Angélica Barril (4)
  Miguel Oscar Giordano (4)
  Gisela Masachessi (4)
  Laura Cecilia Martínez (4)
  María Beatriz Isa (4)
  María Cecilia Freire (6)
  Gabriela López Riviello (5)
  Daniel Cisterna (6)
  Silvia Viviana Nates (4)
  Viviana Andrea Mbayed (3)
  
  1. INIQUI-CONICET, Universidad Nacional de Salta, Av. Bolivia 5150, Salta, 4400, Argentina
  2. Fogarty International Center, University of California at Davis, One Shields Av., Davis, CA, 95616, USA
  3. Cátedra de Virología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 4to piso, Ciudad Autónoma de Buenos Aires, 1113, Argentina
  4. Instituto de Virología Dr. J. M. Vanella, Universidad Nacional de Córdoba, Córdoba, Argentina
  6. Instituto Nacional de Enfermedades Infecciosas INEI-ANLIS Dr. Carlos G. Malbrán, Velez Sarsfield 563, Ciudad Autónoma de Buenos Aires, 1281, Argentina
  5. Departamento Científico Pericial, Prefectura Naval Argentina, Benjamin Lavaisse 1058, Ciudad Autónoma de Buenos Aires, 1107, Argentina
  
• ISSN：1573-2959

文摘

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-.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?) and low concentrations (LC, 104 pfu ml?) of PP7. Tangential ultrafiltration proved to be more efficient (50.36?±-2.91, 17.21?±-.22 and 12.58?±-.35?% for HC in PBS and two river samples, respectively) than adsorption–elution with negatively charged membranes (1.00?±-.34, 2.79?±-.62 and 0.05?±-.08?% for HC in PBS and two river samples, respectively) and polyethylene glycol precipitation (15.95?±-.43, 4.01?±-.12 and 3.91?±-.54?%, for HC in PBS and two river samples, respectively), being 3.2-0.4 times more efficient than the others for PBS and 2.7-52 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.