RNA extraction from virus-diseased sweet potato for reverse transcriptase polymerase chain reaction analysis.

Abstract

Sweet potato virus disease (SPVD), which causes severe crop losses in Africa, is caused by a complex of sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Extraction of pure RNA (for diagnosis by reverse transcriptase polymerase chain reaction [RT-PCR] methods) from diseased sweet potato proved to be difficult using five different total-RNA extraction procedures: (a) that of Chomczynski and Sacchi (1) (b) an adaptation of Lodhi et al., (2) and three commercially available kits (c) Clonsep (Clontech, Clontech Labs Inc, Palo Alto, CA; (d) RNeasy (Qiagen, West Sussex, UK); and (e) RNA isolator (Genosys Biotechnologies). Four of these methods (b-e) generated sufficient RNA, but it was unsuitable for RT-PCR amplification of SPFMV. When these RNA samples were treated with Wizard DNA extraction resin (Promega), the inhibitors of RT-PCR were consistently removed from three (b-d) of these four samples. Another deproteinating step was needed to allow RT-PCR amplification of sample e. The Wizard DNA column purification in conjunction with one of these total RNA extraction methods facilitates quick and reliable extraction of pure RNA for diagnostic purposes and might be suitable for similar problematic plant material.