Detection of Bacillus spores using PCR and FTA filters.
Detection of Bacillus spores using PCR and FTA filters.
复制标题
使用 PCR 和 FTA 过滤器检测芽孢杆菌孢子。
DOI：
10.4315/0362-028x-67.5.1036
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发表时间：
2004
影响因子：
2
通讯作者：
P. Orlandi
中科院分区：
文献类型：
--
作者：
K. Lampel;Deanne Dyer;L. Kornegay;P. Orlandi
重点放在开发和实施微生物病原体快速检测系统上。我们探索了扩展FTA过滤技术在从细菌孢子中制备PCR模板DNA中的应用。将枯草芽孢杆菌、蜡样芽孢杆菌和巨型芽孢杆菌的分离孢子应用于FTA过滤器，并通过PCR扩增出特异性DNA产物。在显微镜下检查孢子制剂，以确保营养细胞的存在，如果有的话，不会产生误导的结果。PCR引物SRM86和SRM87针对细菌rRNA基因的保守区域，而引物Bsub5F和Bsub3R扩增了枯草芽孢杆菌rRNA基因的保守序列的产物。使用后一组引物进行巢式PCR，提高了PCR检测的灵敏度。第一轮PCR检测到53个孢子，巢式PCR检测到5个孢子。FTA过滤器是去除PCR抑制剂的一个很好的平台，在环境、临床和食品样品中具有普遍的应用。
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.