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b>IMPROVEDPCRDETECTIONOFERWINIA CAROTOVORASUBSP.ATROSEPTICA FOR COMMERCIAL USE U HYMAN, PRJ BIRCH and 1K TOTH
IMPROVED PCR DETECTION OF ERWINIA CAROTOVORA SUBSP.ATROSEPTICA FOR COMMERCIAL USE U HYMAN, PRJ BIRCH and 1K TOTH Fungal and Bacterial Plant Pathology, Scottish Crop Research Institute, Invergowne, Dundee. DD2 5DA, UK Background and objectivesErwinia carotovora subsp. atroseptica (Eca). Disease control is based on the production of pathogen free seed tubers, although contamination and latent infection are a constant risk. It is important, therefore, to detect and eliminate contaminated stocks, and particularly those with high levels of latent infection, early in the seed multiplication process. Recently, several Ecaspecific PCR-based assays have been produced to determine Eca contamination of potato stocks. However, these assays are laborious, requiring complex DNA extraction procedures to remove substances in tuber peel extracts which inhibit PCR, and fail to quantify the level of Eca present. If PCR-based assays are to succeed on a commercial scale, both these requirements need to be addressed. This paper describes; i) a simple pre-PCR treatment which avoids complex DNA extraction and, at the same time, increases detection sensitivity, ii) PCR quantification using a competitor DNA template. Materials and methods i) Dilutions of peel extract, naturally contaminated or containing known numbers of Eca, were plated onto crystal violet pectate (CVP) medium and incubated at 270C for 24 h. The resulting bacterial micro-colonies were suspended in water with a glass rod spreader, diluted 1/10 and washed once, before being used in an Eca-specific PCR assay using primers ECAIf and EC~r [1]. The method was validated and the sensitivity confirmed relative to two different commonly used Eca detection methods (CVP, IFC) using naturally contaminated tubers. ii) The amplification product from an Ecaspecific PCR reaction [1], was used to make a competitor template. This template was cloned into pUT miniTn5, and a single copy integrated into the chromosome of an Escherichia coli strain via transposition (E. colieca). Known numbers of E. colieca (103 and 105 cfu ml-1) were added to peel extract containing known numbers of Eca cells or to peel extract from naturally contaminated tubers. Following DNA extraction, the ratio of amplified products from Eca and the competitor template allowed a semiquantitiative assessment of the number of Eca cells in a sample. Results and conclusions i) A simple and sensitive method was developed to replace the need for complex and laborious DNA extraction to remove inhibitory substances in potato tuber peel extract before detection of Eca by PCR [2]. Eca was enriched by a factor of 105 when peel extract was inoculated onto CVP. The sensitivity of detection obtained was ca. 101 cells ml-1, compared to ca. I 03-1 using conventional DNA extraction [1]. The method is simple and cheap, requiring little equipment and reagents, and takes I h of actual work in contrast to 4 h or more with most DNA extraction procedures to carry out 24 sample preparations. Finally, the method allows detection of live Eca cells only which other PCR-based methods and some immunological methods fail to do. ii) Using the competitor template an estimation of Eca cell numbers in peel extract was made. Initially, DNA dilutions of the competitor template were added to the PCR reactions and co-amplified with DNA extracted from peel. This method was then improved when E. colieca was added directly to peel extract before DNA extraction. In addition to the semi-quantification of Eca, the direct addition of E. coli eca cells to peel extract acted as a DNA extraction control, since the ratio between Eca and E. coli eca DNA remained constant regardless of DNA extraction efficiency. The method, which requires a single additional step to conventional PCR, is both simple and accurate. We hope to be able to combine both approaches outlined above for rapid PCR-based quantification of Eca in peel extract. References 1. De Boer SH, Ward U, 1995. Phytopathology 85, 854-858. 143-147.2. Hyman U, Dewasmes V, Toth 1K, Perombelon MCM, 1997. Letters in Applied Microbiology 25,