Comparative Analysis of Three Different Total Nucleic Acid Extraction Protocols for the Diagnosis of Geminiviruses in Squash (Cucurbita moschata)

Abstract

Squash (Cucurbita moschata) is one of the most important crops in tropical countries. Geminiviruses are an important group of plant pathogens. In 2002 a new begomovirus was reported to naturally infect squash and some other crops in Costa Rica. Our objective was to compare, using molecular techniques, the extraction and further purification of DNA from squash by different extraction protocols and storage methods. A single infected sample was collected, half of the material was stored frozen at ) -70C, and the remainder was stored dehydrated in silica gel (SG). Total nucleic acids (TNAs) were extracted by three different protocols and were quantified by fluorometry, and the quality was analysed by electrophoresis in agarose gels, polymerase chain reaction (PCR) of the virus genome, dot blot and Southern blot hybridization. Even though the tissue stored in SG yielded a higher amount of TNAs, the genetic material exhibited lower integrity and this made it useful exclusively for the detection of geminiviral DNA by PCR amplification of short viral sequences and by hybridization with short viral probes. The Dellaporta method proved to be the most effective for the detection of geminiviral DNA in infected squash tissue. Although the cetyltrimethylammonium bromide method showed similar results, the procedure is more time-consuming. Surprisingly, the citrate method showed either similar or worse results than the other methods.