Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay.
Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay.
复制标题
使用集成细胞培养聚合酶链反应 (ICC-PCR) 测定法对牛奶中的活伯氏柯克斯体进行定量。
DOI：
10.1017/s0022029915000400
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Schlesser,Joseph
中科院分区：
文献类型：
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作者：
Stewart,Diana;Shieh,Y-Carol;Tortorello,Mary;Kukreja,Ankush;Shazer,Arlette;Schlesser,Joseph
伯氏柯克斯体细胞内致病菌长期以来一直被认为是原料奶中最耐热的病原体，使其成为确定奶制品巴氏杀菌条件的参考病原体。新的牛奶配方和新的非加热工艺需要验证有效性，这需要比使用目前使用的动物模型来评估柯克斯体存活率更实用的分析方法。此外，人们有兴趣更好地表征各种牛奶配方中柯克斯体的热灭活。为了避免使用豚鼠模型来评估柯克斯体的存活，建立了一种检测牛奶中柯克斯体活性的综合细胞培养-聚合酶链式反应(ICC-PCR)方法。Vero细胞培养物直接从柯克斯体污染的牛奶中感染，放在两个24孔板中。从感染后(第0天)的基础水平(第0天)开始，≥0.5对数基因组当量(GE)/毫升的IS111a基因数量在繁殖后9-11天的水平上显示出奶中柯克斯体的存活能力。脱脂、2%和全脂牛奶中的柯克斯体，以及对半成功感染Vero细胞的Coxiella，使用48小时的感染期和9天的繁殖时间，至少增加了2个对数。在优化的ICC-PCR方法中，全脂牛奶中只有125个柯克斯氏菌/毫升感染和繁殖至少2个对数，但对繁殖的可变确认低至25个柯克斯体/毫升。在MPN格式中，ICC-PCR方法进一步证明了该方法的适用性，以定量60°C热处理0、20、40、60和90分钟后全脂牛奶中剩余的活柯克斯菌数量。
The obligate intracellular pathogen Coxiella burnetii has long been considered the most heat resistant pathogen in raw milk, making it the reference pathogen for determining pasteurisation conditions for milk products. New milk formulations and novel non-thermal processes require validation of effectiveness which requires a more practical method for analysis than using the currently used animal model for assessing Coxiella survival. Also, there is an interest in better characterising thermal inactivation of Coxiella in various milk formulations. To avoid the use of the guinea pig model for evaluating Coxiella survival, an Integrated Cell Culture-PCR (ICC-PCR) method was developed for determining Coxiella viability in milk. Vero cell cultures were directly infected from Coxiella-contaminated milk in duplicate 24-well plates. Viability of the Coxiella in milk was shown by a ≥0·5 log genome equivalent (ge)/ml increase in the quantity of IS111a gene from the baseline post-infection (day 0) level after 9–11 d propagation. Coxiella in skim, 2%, and whole milk, and half and half successfully infected Vero cells and increased in number by at least 2 logs using a 48-h infection period followed by 9-d propagation time. As few as 125 Coxiella ge/ml in whole milk was shown to infect and propagate at least 2 logs in the optimised ICC-PCR assay, though variable confirmation of propagation was shown for as low as 25 Coxiella ge/ml. Applicability of the ICC-PCR method was further proven in an MPN format to quantitate the number of viable Coxiella remaining in whole milk after 60 °C thermal treatment at 0, 20, 40, 60 and 90 min.