Herman L M, De Block J H, Waes G M
Government Dairy Research Station, Agricultural Research Centre Ghent, Melle, Belgium.
Appl Environ Microbiol. 1995 Dec;61(12):4141-6. doi: 10.1128/aem.61.12.4141-4146.1995.
A direct detection method for Clostridium tyrobutyricum spores in up to 100 ml of raw milk is presented. The bacterial spores are concentrated by centrifugation after chemical extraction of the milk components. The vegetative cells are selectively lysed, and their DNA is digested and washed away. Afterwards, the DNA is liberated from the spores by microwave treatment. For the identification of the C. tyrobutyricum DNA, a two-step PCR method with two nested pairs of primers is used. The primers were derived from the 16S-23S rRNA spacer region of C. tyrobutyricum, and the specificity of each of them for C. tyrobutyricum is demonstrated. The detection limit can be estimated to be between 3 and 30 spores in 100 ml of raw milk.
本文介绍了一种直接检测高达100毫升原料乳中酪丁酸梭菌孢子的方法。在对乳成分进行化学提取后，通过离心浓缩细菌孢子。选择性裂解营养细胞，并将其DNA消化并洗去。之后，通过微波处理从孢子中释放DNA。为了鉴定酪丁酸梭菌DNA，使用了一种带有两对嵌套引物的两步PCR方法。引物来源于酪丁酸梭菌的16S - 23S rRNA间隔区，且证明了它们各自对酪丁酸梭菌的特异性。估计在100毫升原料乳中的检测限为3至30个孢子。
