Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR.
Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.
Singh
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Potato Research Centre, Agriculture and Agri-Food Canada, PO Box 20280, NB, E3B 4Z7, Fredericton, Canada. singhr@em.agr.ca
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Singh
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Duplessis
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D005419:Flavonoids; D010636:Phenols / Q000037:antagonists & inhibitors*; D010936:Plant Extracts / Q000737:chemistry; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D011108:Polymers; D059808:Polyphenols; D017800:Potyvirus / Q000235:genetics / Q000302:isolation & purification; D027861:Prunus / Q000737:chemistry* / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D011198:Solanum tuberosum / Q000737:chemistry* / Q000821:virology; D013447:Sulfites / Q000494:pharmacology*
D005419:Flavonoids; D010636:Phenols; D010936:Plant Extracts; D011108:Polymers; D059808:Polyphenols; D012367:RNA, Viral; D013447:Sulfites; C025026:sodium sulfite