PCR detection of Bacillus thuringiensis toxin genes in the environment

The distribution of Bacillus thuringiensis toxin genes in natural samples was determined by polymerase chain reaction amplification of extracted DNA. Two methods of DNA extraction were developed, the first involved direct lysis of cells and spores using a bead beater followed by DNA purification, the second allowed germination of spores in nutrient media and lysis by chemical treatments prior to purification. Detection of B. thuringiensis toxins was performed using a combination of 18 primer pairs that allowed the detection of Lepidopteran active genes (12 pairs), Dipteran active genes (2 pairs) and Coleopteran active genes (4 pairs). From DNA isolated from soil only coleopteran active genes were detected, these were found in 4 of the 12 different soils sampled. The products were cloned and sequence analysis indicated 81-95% similarity to the coleopteran active cryIIIA gene. From leaf samples five of the nine samples taken were positive for either Lepidopteran or Dipteran active toxins, sequence analysis confirmed each product as similar to known B. thuringiensis toxin genes (75-100% similarity). To confirm that this method was appropriate for the detection of B. thuringiensis in the environment, soil samples that had been inoculated with a known B. thuringiensis strain in previous years were sampled. These results have further confirmed that this method was capable of detecting B. thuringiensis toxin genes in natural samples. The spatial location and distribution of toxin genes in natural bacterial populations using this method is under investigation.