Loop-mediated isothermal amplification assay for sensitive and rapid detection of Phytophthora capsici

摘要

Phytophthora capsici is an oomycete plant pathogen that infects peppers, tomatoes, and other agronomic and ornamental crops of the Solanaceae and Cucurbitaceae families worldwide. Early and accurate detection of P. capsici is essential for controlling this disease in the field. The internal transcribed spacer (ITS) regions of nuclear-encoded ribosomal DNA genes are widely used to detect Phytophthora nicotianae, Pythium helicoides and Peronospora variabilis. In this study, a novel and highly sensitive loop-mediated isothermal amplification (LAMP) assay was developed for the specific detection of P. capsici using calcein as a fluorescent indicator. Four LAMP primers were designed based on the ITS sequence of P. capsici. A total of 23 isolates of P. capsici from geographically distinct counties in China yielded positive results in the LAMP assay. No cross-reaction was observed with other oomycetes or fungal pathogens. The detection limit of P. capsici by LAMP was 100fg genomic DNA per 25L reaction. The LAMP assay developed in this study is simple, fast, sensitive and specific, and can be used in the field to detect P. capsici in infected plant tissue.