Food Sensing: Aptamer-Based Trapping of Bacillus cereus Spores with Specific Detection via Real Time PCR in Milk
Food Sensing: Aptamer-Based Trapping of Bacillus cereus Spores with Specific Detection via Real Time PCR in Milk
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DOI：
10.1021/acs.jafc.5b03738
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发表时间：
2015-09-16
影响因子：
6.1
通讯作者：
Fischer, Markus
中科院分区：
文献类型：
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作者：
Fischer, Christin;Huenniger, Tim;Fischer, Markus
需氧孢子给食品制造商和消费者都带来了严重的问题。牛奶尤其面临风险，因此是预防性消费者保护和质量保证的重要问题。芽孢杆菌蜡样芽孢杆菌是一种引起食物中毒的革兰氏阳性病原体，主要产生两种不同类型的毒素：腹泻毒素和催吐毒素。需要可靠、快速的分析方法来检测蜡样芽胞杆菌孢子，这可以通过将体外产生的适体与高度特异性的分子生物学技术相结合来实现。为了开发常规生物分析方法，已经通过表面等离子共振 (SPR) 光谱和荧光显微镜对现有的与蜡样芽胞杆菌孢子具有高亲和力的适体的解离常数和选择性进行了表征。测定了低纳摩尔范围（5.2 至 52.4 nM）的解离常数。随后，利用表征的适体在牛奶模拟缓冲液和脂肪含量在0.3%至3.5%之间的牛奶中建立和验证基于适体的捕获技术。从而，可以实现高达6倍的富集因子。可以看出，捕获方案和表征的适体完全适合在牛奶中的应用。由于适体选择性有限，捕获后采用高度特异性的实时 PCR 测定来获得更高程度的选择性。
Aerobic spores pose serious problems for both food product manufacturers and consumers. Milk is particularly at risk and thus an important issue of preventive consumer protection and quality assurance. The spore-former Bacillus cereus is a food poisoning Gram-positive pathogen which mainly produces two different types of toxins, the diarrhea inducing and the emetic toxins. Reliable and rapid analytical assays for the detection of B. cereus spores are required, which could be achieved by combining in vitro generated aptamers with highly specific molecular biological techniques. For the development of routine bioanalytical approaches, already existing aptamers with high affinity to B. cereus spores have been characterized by surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in terms of their dissociation constants and selectivity. Dissociation constants in the low nanomolar range (from 5.2 to 52.4 nM) were determined. Subsequently, the characterized aptamers were utilized for the establishment and validation of an aptamer-based trapping technique in both milk simulating buffer and milk with fat contents between 0.3 and 3.5%. Thereby, enrichment factors of up to 6-fold could be achieved. It could be observed that trapping protocol and characterized aptamers were fully adaptable to the application in milk. Due to the fact that aptamer selectivity is limited, a highly specific real time PCR assay was utilized following trapping to gain a higher degree of selectivity.