title: Molecular methods (digital PCR and real-time PCR) for the quantification of low copy DNA of Phytophthora nicotianae in environmental samples.
abstract: BACKGROUND
  Currently, real-time polymerase chain reaction (qPCR) is the technique most often used to quantify pathogen presence. Digital PCR (dPCR) is a new technique with the potential to have a substantial impact on plant pathology research owing to its reproducibility, sensitivity and low susceptibility to inhibitors. In this study, we evaluated the feasibility of using dPCR and qPCR to quantify Phytophthora nicotianae in several background matrices, including host tissues (stems and roots) and soil samples.
  
  
  RESULTS
  In spite of the low dynamic range of dPCR (3 logs compared with 7 logs for qPCR), this technique proved to have very high precision applicable at very low copy numbers. The dPCR was able to detect accurately the pathogen in all type of samples in a broad concentration range. Moreover, dPCR seems to be less susceptible to inhibitors than qPCR in plant samples. Linear regression analysis showed a high correlation between the results obtained with the two techniques in soil, stem and root samples, with R(2) = 0.873, 0.999 and 0.995 respectively.
  
  
  CONCLUSIONS
  These results suggest that dPCR is a promising alternative for quantifying soil-borne pathogens in environmental samples, even in early stages of the disease.
authors.affiliation: Department of Soil and Water Conservation and Organic Wastes Management, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Espinardo, Murcia, Spain.
authors.forename: Josefa
authors.initials: J
authors.lastname: Blaya
authors.affiliation: Department of Soil and Water Conservation and Organic Wastes Management, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Espinardo, Murcia, Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: Lloret
authors.affiliation: Department of Soil and Water Conservation and Organic Wastes Management, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Espinardo, Murcia, Spain.
authors.forename: Ana B
authors.initials: AB
authors.lastname: Santísima-Trinidad
authors.affiliation: Department of Soil and Water Conservation and Organic Wastes Management, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Espinardo, Murcia, Spain.
authors.forename: Margarita
authors.initials: M
authors.lastname: Ros
authors.affiliation: Department of Soil and Water Conservation and Organic Wastes Management, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Espinardo, Murcia, Spain.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Pascual
mesh_terms: D004247:DNA / Q000235:genetics*; D004777:Environment; D005240:Feasibility Studies; D018628:Gene Dosage; D010838:Phytophthora / Q000235:genetics* / Q000302:isolation & purification; D031701:Piper; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012987:Soil
chemical_list: D012987:Soil; D004247:DNA
keywords: Phytophthora nicotianae; digital PCR; environmental samples; inhibition; real-time PCR