Detection and quantification of Fusarium oxysporum f. sp. cucumerinum in environmental samples using a specific quantitative PCR assay

The rapid and reliable identification and quantification of pathogens is essential for the management of economically important plant diseases. Fusarium oxysporum f. sp. cucumerinum is the soil borne fungus responsible for Fusarium vascular wilt of cucumber. In this study, we report the development of a specific and reliable real-time quantitative PCR assay and the development of an ultra-sensitive diagnostic pseudo-nested PCR assay. The capacity of the PCR assays to accurately identify and quantify Fusarium oxysporum f. sp. cucumerinum was experimentally tested by the development of standard curves from serial dilutions of copy numbers in a range of complex environmental DNA samples. The amplification efficiency, sensitivity and reproducibility of the qPCR assays were not significantly affected by the presence of any of the non-target background DNA tested. In quantitative real-time PCR, as few as 100 copies could be reliably quantified, and in simple and pseudo-nested PCR as little as 10 pg and 10 fg, respectively, could be detected. This rapid and sensitive qPCR method can be used to facilitate investigations into plant–pathogen interactions, epidemiology, and disease management practices.     

Precautions for Workers

Radioactive isotopes are now available from Chalk River for use by Canadian biologists. Experience has shown that the handling of radioactive isotopes may involve health hazards unless adequate precautions are taken. The nature of these hazards and the type of precautions which must be taken when working with radioactive isotopes are considered. Successful work with radioactive isotopes other than in the smallest tracer amounts requires the use of laboratories and equipment especially designed for the purpose and this is dealt with briefly. The operation of a radioactive laboratory requires certain auxiliary equipment and services, such as health instruments, film monitoring, special laboratory clothing, special cleanable surfaces and disposal of radioactive waste materials. These topics are discussed briefly. Handling of radioactive isotopes involves certain special precautions and a few of these, such as protection of hands, cleaning of glassware, handling of solutions, etc. are reviewed. In addition to protecting all personnel in a laboratory from harmful amounts of radiation, it is necessary to keep the laboratory and the building in which it is housed as free as possible from radioactive substances and this important fact has been stressed.