An improved method for the extraction of nucleic acids from plant tissue without grinding to detect plant viruses and viroids

Abstract

Gene amplification techniques such as polymerase chain reaction (PCR) are widely used for the diagnosis of plant diseases caused by viruses and viroids. It is preferable that sample prep-aration methods for PCR or reverse transcription (RT) PCR are rapid, straightforward, and inex-pensive. We previously reported a method for the extraction of nucleic acids without mechanical tissue grinding using a buffer containing potassium ethyl xanthogenate (PEX) to detect viroid RNAs. In the present report, the previous PEX method was improved and simplified. In the simplified PEX (SPEX) method, the process of PEX buffer treatment for plant cell wall disruption is improved to one step of incubation at 80 °C for 10 min, instead of three steps that took more than 26 min at 65 °C in the previous method. Total nucleic acids could be extracted from fresh, frozen, or dried leaves of a cultivar or wild species of tobacco, tomato, citron, hop plants, and pericarps of persimmon fruits by the SPEX method. Several RNA viruses and viroids were successfully de-tected from the extracted nucleic acids together with an internal mRNA by RT-PCR. The SPEX method may be useful for detecting not only viruses and viroids, but also other plant pathogens.