A five-minute DNA extraction method for expedited detection of Phytophthora ramorum following prescreening using Phytophthora spp. lateral flow devices

• Published in: Journal of microbiological methods Vol. 81; no. 2; pp. 116 - 120
• Main Authors: Tomlinson, J.A., Dickinson, M., Hobden, E., Robinson, S., Giltrap, P.M., Boonham, N.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.05.2010; Elsevier
• Subjects: Biological and medical sciences; Diagnostic sensitivity; Diagnostic specificity; DNA - isolation & purification; DNA isolation; Filtration - methods; Fundamental and applied biological sciences. Psychology; Microbiological Techniques - methods; Microbiology; Phytophthora; Phytophthora - genetics; Phytophthora - isolation & purification; Plant extracts; Polymerase Chain Reaction; Real-time PCR; Sensitivity and Specificity; Sudden oak death; Time Factors; Diagnostic sensitivity; Sudden oak death; Diagnostic specificity; Real-time PCR; DNA isolation; Cardiocirculatory arrest; Sudden death; Cardiovascular disease; Method; Real time; Polymerase chain reaction; Sensitivity; Specificity; Isolation; Detection
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2010.02.006

In a direct comparison with established methods for Phytophthora ramorum detection (isolation followed by morphological identification, or conventional DNA extraction followed by TaqMan real-time PCR) a rapid, simplified detection method in which membranes of lateral flow devices (LFDs) are added directly to TaqMan real-time PCR reactions was used to test 202 plant samples collected by plant health inspectors in the field. P. ramorum prevalence within the 202 samples was approximately 40% according to routine testing by isolation or TaqMan real-time PCR. The diagnostic sensitivity and specificity of the rapid detection method were 96.3% and 91.2%, respectively. This method can be used in conjunction with Phytophthora spp. lateral flow devices to reduce the number of samples requiring testing using more laborious conventional methods. The effect of combining prescreening for Phytophthora spp. with P. ramorum-specific tests is discussed in terms of the positive and negative predictive values of species-specific detection when testing samples collected in different inspection scenarios.