The effect of plant genomic DNA on the detection of mixed infections of begomoviruses using PCR with degenerate primers. (16)
Keywords:Diagnosis, Geminivirus, Multiple infections
AbstractThe feasibility of using polymerase chain reaction (PCR) technology for detecting mixed infections of begomoviruses was assessed in two phases of experiments. First, experiments were conducted to determine the detection limits of single virus templates and of simulated mixtures of two begomoviruses using various concentrations and ratios. A visual detection limit of 500 fg uL-1 was obtained for each virus when used as single viral templates. Simulated mixtures with a 2X, 4X, 8X, and 16X excess of one viral template produced a weakening in band intensity of the less frequent virus though detection was not totally suppressed. Second, experiments were carried out to determine the effect of plant genomic deoxyribonucleic acid (DNA) on the detection of single viral templates and on simulated mixtures. Inhibition of detection of Potato yellow mosaic Trinidad virus (PYMTV) and Tomato leaf curl virus (ToLCV) as single templates began at 100 ng genomic DNA. For simulated mixtures, amplification of the less frequent virus was inhibited at 250 ng or more of genomic DNA. The implications of these findings as well as possible recommendations for improving the sensitivity of PCR towards studying mixed infections of begomoviruses are discussed.