基于Rti-LAMP非预增菌检测肉品中沙门氏菌活菌的关键技术研究

Salmonella enterica (S.enterica) is one of the most common food-borne pathogens. Food-borne disease caused by Salmonella represents a worldwide public health problem. Salmonellosis is most often attributed to the consumption of contaminated foods such as poultry, beef and pork. Typically, ≥100000 Salmonella cells can cause infection in humans, and as few as 15 to 20 organisms are capable of causing Salmonellosis in highly susceptible hosts. . To date, reference methods for bacterial detection or identification rely mainly on culture-based approaches.Traditional culture-based methods for detecting Salmonella are reliable but labor-intensive, too time-consuming, demanding several days for a definitive result, and costly to meet food safety control. On the other hand, molecular methods such as PCR and real-time PCR have been successfully used to rapidly detect a number of food-borne bacterial pathogens including Salmonella in recent years. However, food products can contain a variety of PCR inhibitors such as protein, enzymes, polysaccharides and other compounds. These inhibitors cannot only limit the accuracy, but also the detection sensitivity of pathogens. The fat content of meat is another major hindrance since bacterial cells can be expected to selectively concentrate in the fat phase. So, selective or non-selective pre-enrichment methods are common necessary which require an incubation time of several hours to 1 d. Furthermore, a major disadvantage of molecular detection is its inability to differentiate between viable and dead cells. The development of rapid non-enrichment methodology to allow molecular methodology detection of low numbers of target viable cells such as Salmonella in meat is needed.. The goal of these proposed studies will be to develop a novel detection system based on real time loop-mediated isothermal amplification (Rti-LAMP) methodology with the use of hydrolyzed soluble starch and coated activated carbon combined DNA binding dye for rapid detection low numbers (≤2 CFU/g) of viable Salmonella in meat within 4 h without enrichment. Firstly, it will be to develop an efficacy of amylose and soluble starch for capturing fat in meat resulting in all bacterial cells forced into the aqueous phase. Then, activated carbon coated with milk proteins and bentonite will be used to adsorb soluble compounds from meat but not to bind targeted bacterial cells. The purified Salmonella cells will be treated with DNA binding dye to differentiate viable and dead cells. Salmonella's invA or other specific genes will be used as target genes to design primers for the Rti-LAMP assays. Finally, the developed detection system will be used to detect Salmonella from market meat samples for evaluation the sensitivity and accuracy. The research will supply a novel method system for the rapid detection of low number of viable Salmonella cells in meat without enrichment.

由沙门氏菌等致病菌引起的食源性疾病是全球公共卫生面临的严峻挑战。为了有效预防致病菌污染产生的食品安全问题，已发展了多种致病菌的快速检测技术。然而，其在应用于食品样品检测时，因受食品组分干扰，一般都需预增菌培养，导致检测过程耗时费力。因此，如何消除食品组分影响，实现非预增菌检测食品中的致病菌是快速检测技术研究的关键。本项目拟以肉品中油脂和水溶性组分的吸附剂制备为基础，以肉品中沙门氏菌的高效回收精制-死活细菌的区分-快速检测技术建立-为主线，重点研究吸附剂前处理肉样的工艺条件及其分子机制，回收精制的细菌样品再经DNA染料处理以区分死、活细胞；联合这些前处理技术，优化检测目的基因的引物，建立实时荧光环介导等温扩增技术检测沙门氏菌，实现4h以内从肉品中检测≤2CFU/g沙门氏菌活菌的目标。预期结果不仅将为肉品中沙门氏菌的快速检测提供一种新方法，而且可为食品中其它致病菌的检测研究提供重要的科学借鉴。

食品中的致病菌快速检测一般存在三个主要困难：一是食品基质的复杂性，、物理状态多样、粘度和均一性差异；二是含有多种杂菌且分布不均匀；三是污染致病菌浓度一般较低，给食品污染的致病菌快速检测带来了巨大挑战。本项目基于Rti-LAMP特异性检测技术，以食品中目标细菌的高效回收精制技术联合鉴别区分死活细菌的EMA方法为关键突破，研制了新型非预增菌快速检测方法，具体为：A. 筛选出有利于酶解吸附油脂的土豆可溶性淀粉，建立和优化了ɑ-淀粉酶水解淀粉的工艺条件，测定了酶解淀粉对油脂的吸附性能及细菌回收效果；B. 以活性炭为食品水溶性成分的吸附剂，蒙脱石/脱脂奶粉为封闭剂，优化了封闭活性炭的制备工艺条件，测定了其用于食品样品处理不吸附细菌但保持吸附食品水溶性DNA聚合酶抑制剂性能，表征了封闭活性炭及其吸附机制；C. 建立了抗体交联磁珠的免疫吸附食品样品前处理技术，不同食品最适的前处理方法或有不同；D. 建立优化了溴化乙锭单叠氮溴（EMA）和单碘化丙锭（PMA）DNA染料区分食品中死、活沙门氏菌的浓度及作用条件；E. 设计扩增沙门氏菌特异目标基因引物，以midori为核酸荧光染料，建立了沙门氏菌、大肠杆菌O157:H7实时环介导等温扩增技术Rti-LAMP。F. 通过集成可溶性淀粉、封闭活性炭的食品样品前处理技术以及核酸螯合剂区分死、活细胞各技术，建立了基于Rti-LAMP快速检测肉类食品中沙门氏菌活菌的方法，实现了约4 h从食品中检测≤2 CFU/g左右沙门氏菌活菌污染检测目标。该方法用于市场肉、奶食品沙门氏菌污染的检测，与国标法检测结果相符率90%以上。本研究为食源性致病菌快速检测提供了新的检测技术，实现了非预增菌快速检测，有望用于食品样品的快速检测、筛查，具有重要的理论意义和应用价值。