Detection of Bacillus spores using PCR and FTA filters.
Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results. PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B. subtilis rRNA gene. With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.
Lampel
Keith A
KA
U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Laurel, Maryland 20708, USA. klampel@cfsan.fda.gov
Dyer
Deanne
D
Kornegay
Leroy
L
Orlandi
Palmer A
PA
D001407:Bacillus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005374:Filtration; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013171:Spores, Bacterial / Q000302:isolation & purification*
D017931:DNA Primers; D004269:DNA, Bacterial