Use of dried high-phenolic laden host leaves for virus and viroid preservation and detection by PCR methods

Abstract

The efficiency of RNA extraction for Apricot latent virus (ApLV), Plum bark necrosis stem pitting associated virus (PBNSPaV), Prunus necrotic ring spot virus (PNRSV), Potato virus Y (PVY), and Apple scar skin viroid (ASSVd) from infected hosts is of great importance for molecular diagnosis by the polymerase chain reaction (PCR). A method is described for drying tissue to overcome phenolic inhibitors of viral RNA. This study showed that the infected host leaves, dried at 65 °C for 2 days and conserved at 4 °C in air proof conditions, serve as good sources for detection of viral and viroid pathogens by PCR methods. Preliminary results suggest that ApLV, PNRSV, PVY, and ASSVd were detected easily by reverse transcriptase-polymerase chain reaction (RT-PCR) and PBNSPaV by nested-RT-PCR with high amplification yields. No significant difference was observed between ethidium bromide-stained band profiles of dried compared to fresh leaves of infected samples. The RNA derived from dry leaf samples was suitable for detection studies. This simple and inexpensive method has proved very effective for long term conservation of virus and viroid isolates.