DEVELOPMENT OF A PCR ASSAY FOR DETECTION OF SPORE‐FORMING BACTERIA

Abstract

Abstract Degenerate PCR primers were designed based on the published nucleotide sequences of the sporulation sigma factor ***oE (spoIIGB) from Bacillus subtilis, Bacillus thuringiensis, and Clostridium acetobutylicum. The primer set 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 Clostridium species, Paenibacillus polymyxa, Thermoanaerobacterium thermosaccharolyticum, Moorella thermoacetica) and in nonspore‐forming bacteria. Under optimized PCR conditions, all spore‐forming bacteria tested yielded a PCR product of the expected size (∼360bp), although the nonspore‐forming Listeria monocytogenes and Lactococcus lactis subsp. lactis also yielded PCR products of this approximate size. To improve the specificity and sensitivity of this assay, we Southern blotted gel electrophoresis‐separated PCR products with a digoxigenin‐labeled B. subtilis spoIIGB probe. This probe hybridized with the ∼ 360 bp PCR product from all spore‐forming species but did not hybridize with PCR products of this approximate size from any nonspore‐forming bacteria. The PCR‐Southern blot assay was 100 to 1,000‐fold more sensitive than PCR alone, yielding a lower detection limit of approximately 3 CFU spore‐forming bacteria/PCR reaction. We conclude that, based on amplicon size and Southern hybridization, this strategy provides a viable approach for detecting spore‐forming bacteria.