title: Application of a simple and affordable protocol for isolating plant total nucleic acids for RNA and DNA virus detection.
abstract: Standard molecular methods for plant virus diagnosis require the purification of RNA or DNA extracts from a large number of samples, with sufficient concentration and quality for their use in PCR, RT-PCR, or qPCR analysis. Most methods are laborious and use either hazardous and/or costly chemicals. A previously published protocol for RNA isolation from several plant species yields high amounts of good quality RNA-DNA mixture in a simple, safe and inexpensive manner. In the present work, this method was tested to obtain RNA-DNA extracts from leaves of tomato, potato and three species of citrus, and was compared with two commercial kits. The results demonstrated that this protocol offers at least comparable nucleic acid quality, quantity and purity to those provided by commercial phenol-based or spin column systems and that are suitable to be used in PCR, RT-PCR and qPCR for virus and viroid detection. Because of its easy implementation and the use of safe and inexpensive reagents, it can be easily implemented to work in plant virus and viroid detection in different plant species.
authors.affiliation: Instituto Nacional de Investigación Agropecuaria, CP 50000, Salto, Uruguay. Electronic address: aarruabarrena@inia.org.uy.
authors.forename: Ana
authors.initials: A
authors.lastname: Arruabarrena
authors.affiliation: Laboratorio de Virología Molecular, Centro Universitario Regional Noroeste (CENUR Noroeste), Universidad de la República, Rivera 1350, 50000, Salto, Uruguay.
authors.forename: María José
authors.initials: MJ
authors.lastname: Benítez-Galeano
authors.affiliation: Instituto Nacional de Investigación Agropecuaria, CP 50000, Salto, Uruguay.
authors.forename: Mario
authors.initials: M
authors.lastname: Giambiasi
authors.affiliation: Instituto Nacional de Investigación Agropecuaria, CP 50000, Salto, Uruguay.
authors.forename: Ana
authors.initials: A
authors.lastname: Bertalmío
authors.affiliation: Laboratorio de Virología Molecular, Centro Universitario Regional Noroeste (CENUR Noroeste), Universidad de la República, Rivera 1350, 50000, Salto, Uruguay.
authors.forename: Rodney
authors.initials: R
authors.lastname: Colina
authors.affiliation: Instituto de Investigaciones en Fruticultura Tropical, 7th Ave. #3005, CP 11300, Playa, La Habana, Cuba.
authors.forename: Lester
authors.initials: L
authors.lastname: Hernández-Rodríguez
mesh_terms: D002957:Citrus / Q000235:genetics / Q000821:virology; D018744:DNA, Plant / Q000302:isolation & purification; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods; D018515:Plant Leaves / Q000235:genetics* / Q000821:virology; D010942:Plant Viruses / Q000235:genetics* / Q000302:isolation & purification*; D010944:Plants / Q000235:genetics* / Q000821:virology; D018749:RNA, Plant / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods* / Q000592:standards; D011198:Solanum tuberosum / Q000235:genetics / Q000821:virology; D014772:Viroids / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D018744:DNA, Plant; D004279:DNA, Viral; D018749:RNA, Plant; D012367:RNA, Viral
keywords: DNA; PCR; Plant viroid; Plant virus; Purification; RNA; RT-PCR; qPCR