Evaluation of DNA extraction methods from dried and frozen tomato leaves for detecting begomoviruses by PCR.

SUMMARY Six DNA extraction protocols for dried and frozen tomato [Solanum lycopersicum (Mill)] leaves were evaluated to detect begomoviruses using polymerase chain reaction (PCR). The extraction method comparisons were based on DNA quantity and quality, extraction cost, and processing time. Four samples of dried and frozen tomato leaves, collected in the Venezuelan states of Zulia, Merida and Tachira, were evaluated. The DNA concentration and the ratios of 260 and 280µm absorbances were measured using spectrophotometry. Begomovirus detection was performed using degenerate primers for viral components A and B (DNA-A and DNA-B), and PCR band intensity was recorded using a visual scale. The SDS method produced greater DNA quantity and quality, and it is faster and less expensive than the other extraction protocols tested. Begomovirus DNAB was more consistent in amplification than DNA-A, sugges ting higher variability of the A component. Resuspended DNA pellets minimized the effects of inhibitors on the amplification because of the high sensitivity of the PCR technique, which also allowed begomovirus detection even when using small amounts of dried and frozen leaf tissue. The DNA isolated by the SDS method provides a simple and economical option for begomovirus screening in tomato using PCR, and could be used for large-scale epidemiological studies.