White spot syndrome virus (WSSV) continues to be the most pathogenic among the penaeid shrimp viruses. In this study, WSSV DNA was detected in pond soil samples using a 2-step nested PCR. Primers described previously were used for the first round of amplification and based on the sequenced amplicon, an inner primer was designed for the 2nd round of amplification. Using plasmid DNA (pET 100) containing the 211 bp target WSSV sequence, analytical sensitivity showed that the 2-step nested PCR protocol was able to detect down to 0.015 fg of the plasmid DNA, or approximately 2 copies of the target DNA sequence. Persistence of WSSV DNA in pond soil samples after various time intervals was determined. WSSV-specific PCR product (161 bp) was still present in the soil samples even after 10 months of storage. The effect of soil heat treatment on the WSSV DNA was also examined. Soils were subjected to 25, 37, 50 and 70 degrees C for 1, 3 and 5 days. The results showed that PCR amplifiable WSSV DNA was still present even after 5 days at 70 degrees C. To our knowledge, this is the first report on the detection of WSSV DNA in soil samples. Based on these findings, it is concluded that the persistence of viral DNA in soil habitats may be an important aspect of WSSV ecology and may have an implication for viral transmissibility.