Title: An expedited method to isolate DNA for PCR from Magnaporthe oryzae stored on filter paper Authors
|Wamishe, Yeshi -|
|Zhou, Bo -|
Submitted to: The Crop Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 3, 2014
Publication Date: October 1, 2014
Repository URL: http://handle.nal.usda.gov/10113/60691
Citation: Jia, Y., Wamishe, Y.A., Zhou, B. 2014. An expedited method to isolate DNA for PCR from Magnaporthe oryzae stored on filter paper. The Crop Journal. 2(5):267-271. Interpretive Summary: Gene detection using polymerase chain reaction (PCR) is a common method for microbial identification and diagnosis. The classical method of fungal DNA preparation for PCR is multi-step and includes growing the fungus in liquid or solid medium, lyophilizing mycelia, disruption of cell walls, removal of proteins with phenol and chloroform, and precipitation of DNA with ethanol or isopropanol. This method is time consuming, labor intensive, and uses phenol and chloroform compounds that result in toxic chemical waste. In the present study, we developed a simple, fast, and inexpensive method for amplification of a gene from the fungal pathogen that causes rice blast disease using fungal samples directly from storage. As a positive control fungal DNA was extracted using classical DNA preparation method. The PCR was performed with DNA at preparation day, and after 4, 8, 10, and 18 days in refrigerated storage. Overall, nearly 90 percent of the samples of DNA prepared directly from fungus on the filter paper appeared suitable for use in further genetic assays. We anticipate that this method will benefit specialists who work on crop breeding and protection greatly and may be easily adopted for many other fungi.
Technical Abstract: The fungus Magnaporthe oryzae is the causal agent for a wide range of cereal diseases. For long-term preservation, the fungus is grown and stored desiccated on filter papers at -20° C. Inoculated filter papers were cut into pieces from 0.5-1 cm diameter prior to storage. In the present study, a quick (11 minutes) and simple method to prepare DNA suitable for amplifying avirulence genes of M. oryzae by polymerase chain reaction (PCR) was developed. A piece of filter paper containing the fungus was removed from a glass bottle and submerged in a 0.2 mL Eppendorf tube containing 100 µL 10 X TE. The suspension was heated for 10 minutes at 95°C using a PCR machine. The tube was then centrifuged for 1 minute at 3000 rpm. One µL of 10 X TE solution containing DNA was used for PCR. A total of 28 samples were PCR tested. As a positive control, fungal DNA was extracted using classical DNA preparation methods. DNA samples obtained from both methods were stored at 4°C. The PCR was performed with DNA at preparation day, and after 4, 8, 10, and 18 days in refrigerated storage. Four samples namely, #12, #13, #14, and #28 failed to amplify AVR-Pi9. These four samples were tested with a different set of primers for AVR-Pi9, and for AVR-Pita1 verifying that the quality of these 4 samples was not good for PCR. Overall, nearly 90 percent (24/28) of the samples of DNA prepared directly from fungus on the filter paper appeared suitable for a rapid survey of genetic identity of the rice blast fungus by PCR. Therefore, this method will be useful and effective in reducing costs and time and could readily be adopted worldwide for analysis of M. oryzae and possibly other fungi.