Cooper Robin M, McKillip John L
Roche Diagnostics Corporation, Indianapolis, IN 46256, USA.
J Basic Microbiol. 2006;46(5):358-64. doi: 10.1002/jobm.200510132.
Dry milk powders and functional ingredients frequently contain high levels of viable bacterial spores, some of which may result in growth of toxigenic Bacillus spp. in reconstituted and temperature-abused foods. Samples from nonfat dry milk (NFDM), infant milk formula (IMF), coffee creamer, lecithin, and cocoa powder were subjected to a short heat treatment followed by enrichment in tryptone phosphate glucose yeast extract (TPGY) broth at 32 degrees C for 12-25 hours to obtain cell densities of 10(6) CFU ml(-1). DNA was extracted using a modification of established protocol, leading to the development of an optimized method for each food system. Purified DNA was amplified by rep-PCR using extragenic sequence-targeting primers and optimized for each food. PCR fingerprints from each food were analyzed electrophoretically for banding patterns earlier correlated to that of enterotoxigenic Bacillus spp. and Bacillus cereus positive control DNA fingerprints. Reverse passive latex agglutination (RPLA) and Bacillus Diarrhoeal Enterotoxin Enzyme Linked Immunosorbent Assay (Tecra Diagnostics) confirmed the presence of HBL and NHE enterotoxin production in NFDM, Coffee creamer, infant milk formula, and two lecithin samples but not in cocoa powder. These results demonstrate the utility of rep-PCR not only as a tool for bacterial genotyping, but a unique means of quality control and hygiene monitoring in food microbiology.
奶粉和功能性成分通常含有大量活的细菌孢子，其中一些可能导致在复原和温度滥用的食品中产毒芽孢杆菌属生长。从脱脂奶粉（NFDM）、婴儿配方奶粉（IMF）、咖啡奶精、卵磷脂和可可粉中采集的样品经过短时间热处理，然后在32℃的胰蛋白胨磷酸盐葡萄糖酵母提取物（TPGY）肉汤中富集12 - 25小时，以获得10(6) CFU ml(-1)的细胞密度。使用改良的既定方案提取DNA，从而为每个食品体系开发出一种优化方法。使用针对基因外序列的引物通过重复聚合酶链反应（rep-PCR）扩增纯化的DNA，并针对每种食品进行优化。对每种食品的PCR指纹图谱进行电泳分析，以检测与产肠毒素芽孢杆菌属和蜡样芽孢杆菌阳性对照DNA指纹图谱相关的条带模式。反向被动乳胶凝集试验（RPLA）和芽孢杆菌腹泻性肠毒素酶联免疫吸附测定（Tecra诊断）证实，脱脂奶粉、咖啡奶精、婴儿配方奶粉和两份卵磷脂样品中存在HBL和NHE肠毒素产生，但可可粉中未检测到。这些结果表明，rep-PCR不仅可作为细菌基因分型的工具，而且是食品微生物学中质量控制和卫生监测的独特手段。
