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 preparation methods for PCR or reverse transcription (RT) PCR are rapid, straightforward, and inexpensive. 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 detected 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.
Highlights
Plant viruses and viroids are responsible for substantial losses in yield and reduce the quality of fruits and flowers in many crops worldwide [1]
Leaf tissue of 0.1–0.3 g was used in the previous potassium ethyl xanthogenate (PEX) method [11], whereas leaf discs with a reduced amount of 0.05–0.1 g was used in the modified simplified PEX (SPEX) method so as to replicate the amount of tissue from each test plant by the number of discs, to transfer plant tissue cut out from leaf to the centrifuge tube using a plastic straw, and to fully immerse the tissue in the PEX buffer for plant cell wall disruption efficiently
The modified SPEX method was conducted by one step of incubation in the same way as Jhingan [9], but it was shortened to 10 min at various temperatures
Summary
Plant viruses and viroids are responsible for substantial losses in yield and reduce the quality of fruits and flowers in many crops worldwide [1]. Diagnosis of viral and viroid diseases is critical for disease control because there are no substantially effective agricultural chemicals that act directly on these pathogens. Polymerase chain reaction (PCR) and reverse transcription (RT) PCR are useful for detecting plant viruses, especially some viruses that are difficult to apply serological detection methods. Viroids are circular, single-stranded RNA molecules known as the smallest plant pathogens and do not encode any proteins [2]; they cannot be detected by serological methods [3]. RT-PCR is widely used for detecting viroids in addition to some plant viruses for disease diagnosis, and in laboratory studies. The application of highly sensitive PCR for diagnostics by the amplification of various target sequences is often hampered by problems of false-positives generated with nucleic acid contamination, e.g., (i) cross-contamination among samples, (ii) carryover contamination from previous amplified products, (iii) laboratory workstation surface contamination, (iv) contamination of laboratory instruments and equipment including micropipettes, tube racks, centrifuge, vortex mixer, (v) contamination in the PCR reagents
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