Abstract

HERNANDEZ GUZMAN, Anngie Katherine  and  GUZMAN-BARNEY, María Mónica. Comparison of RNA isolation methods for Potato yellow vein virus (PYVV) detection by RT-PCR in different organs of Solanum tuberosum Group Phureja. Rev. colomb. biotecnol [online]. 2013, vol.15, n.1, pp.71-81. ISSN 0123-3475.

Potato yellow vein virus (PYVV, Crinivirus/Closteroviridae), contains a tripartite genome with ssRNA(+), is phloem limited and can affect yield reduction. PYVV is a re-emergent virus and is a quarantine pathogen in Europe and United States. The detection has been based in RT-PCR, NASH and real time RT-PCR (RT-qPCR) in leaflets and tuber shoots samples. It is not known how is the distribution of PYVV within infected plants, for that is necessary to establish an efficient RNA isolation method for obtaining material from different organs. The objective of the present work was to evaluate three RNA isolation methods: a Trizol® (Invitrogen) based method, a method using hexadecyltrimethylammonium bromide (CTAB) and the phenol - chloroform method followed by Sephadex columns purification; for virus detection using RT-PCR in: leaflets, petiole, peduncle, petals, stem, aerial and subterranean roots, anther and shoot tuber; of infected plants. The isolation methods were tested in terms of integrity (ratio of band intensity of 28S and 18S ribosomal subunits), quality (absorbance 260/280 ratio) and total yield. PYVV was detected by RT-PCR in all organs analyzed by the three isolation methods. There were no significant differences found among the three isolation methods, although, Trizol® (Invitrogen) presented high yield, quality and integrity, furthermore Trizol® (Invitrogen) allowed the virus detection using RT-PCR in all analyzed organs.

Keywords : Crinivirus; PYVD; Trizol®; CTAB; Sephadex.

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