Development of a PCR assay for detection of spore-forming bacteria

Degenerate PCR primers were designed based on the published nucleotide sequ ences of the sporulation sigma factor sigma(E) (spoIIGB) from Bacillus subt ilis, Bacillus thuringiensis, and Clostridium acetobutylicum. The primer se t was used in a Hot Start Touch Down-PCR to screen for the presence of the target gene in both spore-forming (eight Bacillus species, eight Clostridiu m species, Paenibacillus polymyxa, Thermoanaerobacterium thermosaccharolyti cum, Moorella thermoacetica) and in nonspore-forming bacteria. Under optimi zed PCR conditions, all spore-forming bacteria tested yielded a PCR product of the expected size (similar to 360 bp), although the nonspore-forming Li steria monocytogenes and Lactococcus lactis subsp. lactis also yielded PCR products of this approximate size. To improve the specificity and sensitivi ty of this assay, we Southern blotted gel electrophoresis-separated PCR pro ducts with a digoxigenin-labeled B. subtilis spoIIGB probe. This probe hybr idized with the similar to 360 bp PCR product from all spore-forming specie s but did not hybridize with PCR products of this approximate size from any nonspore-forming bacteria. The PCR-Southem blot assay was 100 to 1,000-fol d more sensitive than PCR alone, yielding a lower detection limit of approx imately 3 CFU spore-forming bacteria/PCR reaction. We conclude that, based on amplicon size and Southern hybridization, this strategy provides a viabl e approach for detecting spore-forming bacteria.