Mercanoglu Taban B, Ben U, Aytac S A
Gazi University, Vocational School of Health Services, Golbasi, Ankara, Turkey.
J Dairy Sci. 2009 Jun;92(6):2382-8. doi: 10.3168/jds.2008-1537.
During the past few years, milk has presented a risk of Salmonella contamination; it has been implicated as the cause in several outbreaks of salmonellosis. Because conventional detection methods require 5 to 7 d for completion and involve several subcultivation stages followed by biochemical and serological tests, rapid and sensitive methods have been sought, mainly at the DNA level. Therefore, a study including milk samples was conducted to evaluate the performance of a combination of 2 techniques--immunomagnetic separation and polymerase chain reaction (PCR)--for the detection of Salmonella. The 16-, 14-, 12-, 10-, and 8-h nonselective pre-enrichment steps before immunomagnetic separation and the high-pure DNA preparation method before PCR were used in a combined assay. Milk samples, which were found to be Salmonella-negative by a reference method, were first inoculated with Salmonella Enteritidis. Next, the shortest pre-enrichment time that is required for detection of 1 or 10 cfu of Salmonella/mL by combined immunomagnetic separation-PCR assay was found by using 16-, 14-, 12-, 10-, and 8-h incubation periods. The detection limit using a 16-, 14-, or 12-h nonselective pre-enrichment was 1 to 10 cfu/mL. However, the sensitivity decreased to 10(1) and 10(2) cfu/mL, respectively, when 10- and 8-h pre-enrichments were used. This assay, in conjunction with a 12-h pre-enrichment, proved to be rapid (overall 16 h) and sensitive (1-10 cfu/mL) for the detection of Salmonella in milk samples and promising for routine use in the detection of Salmonella in milk.
在过去几年中，牛奶存在沙门氏菌污染风险；它已被认定为几起沙门氏菌病疫情的病因。由于传统检测方法需要5至7天才能完成，且涉及多个传代培养阶段，随后还要进行生化和血清学检测，因此人们一直在寻找快速且灵敏的方法，主要是在DNA水平上。因此，开展了一项包含牛奶样本的研究，以评估免疫磁珠分离和聚合酶链反应（PCR）这两种技术联用对沙门氏菌的检测性能。在联用检测中，采用了免疫磁珠分离前16、14、12、10和8小时的非选择性预富集步骤以及PCR前的高纯DNA制备方法。首先用肠炎沙门氏菌接种经参考方法检测为沙门氏菌阴性的牛奶样本。接下来，通过16、14、12、10和8小时的培养期，找出联用免疫磁珠分离 - PCR检测法检测每毫升牛奶中1或10个沙门氏菌菌落形成单位（cfu）所需的最短预富集时间。使用16、14或12小时非选择性预富集时的检测限为1至10 cfu/mL。然而，当采用10小时和8小时预富集时，灵敏度分别降至10¹和10² cfu/mL。该检测法结合12小时预富集，被证明对牛奶样本中沙门氏菌的检测快速（总共16小时）且灵敏（1 - 10 cfu/mL），有望用于牛奶中沙门氏菌检测的常规操作。
