Simple and rapid preparation of infected plant tissue extracts for PCR amplification of virus, viroid, and MLO nucleic acids.
A rapid, simple method for preparing plant tissues infected with viruses, viroids, or MLOs using a commercial product known as Gene Releaser is described. The Gene Releaser polymeric matrix method produced plant extracts suitable for PCR amplification without the use of organic solvents, ethanol precipitation, or additional nucleic acid purification techniques. Modification of maceration methods and/or extraction buffers resulted in the PCR amplification of potato spindle tuber, apple scar skin, and dapple apple viroids, as well as, genomic segments of plum pox potyvirus, grapevine virus B, grapevine leafroll-associated virus III, and elm yellows MLO. These pathogens were amplified from tissue of woody and herbaceous hosts such as peach, apricot, apple, grapevine, elm, periwinkle and potato. The application of this product for use with intractable tissue avoids lengthy and laborious extraction procedures. In our hands, about 20 samples could be prepared for PCR or RT-PCR in 1-2 h versus 1-3 days.
Levy
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U.S. Department of Agriculture, National Germplasm Resources Laboratory, Beltsville, MD 20705-2350.
Lee
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Hadidi
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D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000235:genetics / Q000302:isolation & purification; D009696:Nucleic Acids / Q000235:genetics* / Q000302:isolation & purification*; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification; D010944:Plants / Q000382:microbiology* / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014772:Viroids / Q000235:genetics / Q000302:isolation & purification; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D004269:DNA, Bacterial; D004279:DNA, Viral; D009696:Nucleic Acids