Jaakohuhta Sinikka, Härmä Harri, Tuomola Mika, Lövgren Timo
Raisio plc, P.O. Box 101, FI-21201 Raisio, Finland.
Int J Food Microbiol. 2007 Mar 20;114(3):288-94. doi: 10.1016/j.ijfoodmicro.2006.09.025. Epub 2006 Dec 13.
Listeria spp. are Gram-positive rod shaped bacteria found universally in the environment. Pathogenic Listeria monocytogenes is seldom harmful to healthy adults, but can cause serious disease, listeriosis, especially to pregnant women, neonates, and elderly or immunocompromised people. Conventional methods for screening Listeria in food samples are time consuming and laborious, involving the use of a range of liquid media and plate cultures. In the current study, the total analysis time was shortened by employing a sensitive Listeria assay, which was able to detect the bacteria in low concentrations. Sensitivity of the sandwich immunoassay was substantially improved by utilizing europium(III)-chelate containing latex nanoparticles as tracers. Each 107 nm nanoparticle contained approximately 31000 europium(III)-chelates which enhanced the specific activity of the label. The sensitive nanoparticulate immunoassay developed for Listeria spp. was performed in one-step and two-step formats. One-step assay was notably faster, 15 min, and simpler to execute having analytical sensitivity of 300 CFU/ml and a dynamic range of three orders of magnitude. The sensitivity, 20 CFU/ml, of the 4 h two-step assay clearly exceeded that of the one-step assay, and the dynamic range was nearly five orders of magnitude. Food and environmental samples were measured against a commercial L. monocytogenes immunoassay with good correlation. The developed sensitive assay enabled shorter sample enrichment times and, therefore, faster analysis of Listeria spp. Obviously the detection of several other bacteria can also be enhanced by applying the nanoparticle assay technology.
李斯特菌属是革兰氏阳性杆菌，普遍存在于环境中。致病性单核细胞增生李斯特菌对健康成年人很少有害，但可导致严重疾病——李斯特菌病，尤其是对孕妇、新生儿以及老年人或免疫功能低下者。食品样品中李斯特菌的传统筛选方法既耗时又费力，需要使用一系列液体培养基和平板培养。在本研究中，通过采用一种灵敏的李斯特菌检测方法缩短了总分析时间，该方法能够检测低浓度的细菌。利用含铕（III）螯合物的乳胶纳米颗粒作为示踪剂，夹心免疫测定的灵敏度得到了显著提高。每个107纳米的纳米颗粒含有约31000个铕（III）螯合物，增强了标记物的比活性。为李斯特菌属开发的灵敏纳米颗粒免疫测定以一步法和两步法进行。一步法明显更快，只需15分钟，执行也更简单，分析灵敏度为300 CFU/ml，动态范围为三个数量级。4小时两步法的灵敏度为20 CFU/ml，明显超过一步法，动态范围近五个数量级。针对食品和环境样品与市售单核细胞增生李斯特菌免疫测定进行测量，具有良好的相关性。所开发的灵敏测定方法能够缩短样品富集时间，因此能够更快地分析李斯特菌属。显然，应用纳米颗粒测定技术也可以提高对其他几种细菌的检测能力。
