Application of molecular methods for detection of Pochonia chlamydosporia from soil.

Two extraction and detection procedures were compared for isolation of the nematode parasitic fungus, Pochonia chlamydosporia , from soil. The extraction methods compared were a traditional, chloroform-based DNA extraction protocol, and the use of nucleic acids captured by magnetic beads. Sensitivity and specificity tests were developed using DNA extracted from a sandy soil treated with a P. chlamydosporia isolate VC21, or from cultures of the same isolate used as a control. The fungus DNA was detected through molecular hybridization (dot-blot) or Real-time PCR. Two probes, of 160 and 197 nucleotides respectively, were synthesized for dot-blot. They recognized an internal region of a serine protease (VCP1) gene, or a fragment of the mitochondrial rRNA gene (NMS). For Real-time PCR a molecular beacon specific to a 23 bp fragment within the VCP1 gene was used. The conventional extraction did not yield DNA pure enough to allow PCR, but permitted its identification through the dot-blot analysis. This protocol allowed the contemporary test of several samples with a detection limit of 500 ng of total DNA from soil. The use of magnetic beads in the DNA extraction from soil simplified the procedure allowing the collection of amounts of high quality DNA, ready for use in Real Time PCR in minutes.