Development of a simple and effective method for specific detection of Pepper mild mottle virus

A digoxigenin (DIG)-labeled cDNA probe complementary to the region from 5,256-6,300 nt of Pepper mild mottle virus (PMMoV) genome was synthesized. The specificity and sensitivity of the probe was tested by the dot-blot hybridization. The detection limit of this method was equivalent to 0.8 microg of fresh infected tissue in each spot. Double-antibody sandwich (DAS) ELISA and RT-PCR had the detection limit 39 microg and 0.008 microg of fresh infected tissue, respectively. We evaluated leaf, fruit pulp, and seed of pepper plant by dot-blot hybridization and found all the tested tissues suitable for detection of PMMoV. Finally, 111 tissue samples including 93 samples collected from the pepper fields of Beijing and Baoding and 18 commercial seed samples were evaluated by this method. The results showed that the incidence rate of the infected samples was 14% and 61% for the field samples and commercial seeds, respectively. The high sensitivity and reliability of the molecular hybridization assay provided an important alternative method for the detection of PMMoV in a large-scale.