食源性致病菌快速检测方法研究进展

食源性致病菌污染是导致食品安全问题的重要因素,食源性致病菌的检测已成为近年来研究的热点.以免疫分析、分子生物学、生物传感器、代谢组学、核酸适配体等技术为基础的快速检测方法发展迅速,已成为检测食源性致病菌的主要方法.该文结合近年来各种快速检测方法的相关研究进展,介绍了以上述技术为基础的快速检测食源性致病菌的方法,并讨论了...     

纳米粒子在食源性致病菌检测中的应用进展

食品安全事关人民群众的身体健康和生命安全,而食源性致病菌是食品安全的主要影响因素。由食源性致病菌引起的疾病和死亡持续威胁着全球的公共卫生安全。因此,开发快速、准确且灵敏的食源性致病菌检测方法是预防食源性疾病暴发和确保食品安全的关键。常规检测方法费时费力,需要昂贵的设备和专业的人员,应用受限。近年来,随着纳米技术的快速发展,纳米粒子凭借其小尺寸、高比表面积和高反应活性等理化特性成为食源性致病菌检测领域的研究热点。此外,将识别元件修饰于纳米粒子表面并结合新颖的分析技术,能提高检测的特异性和灵敏度。该综述主要总结和比较了磁性纳米粒子、贵金属纳米粒子、荧光纳米粒子和二氧化硅纳米粒子在食源性致病菌检测中的应用,以期为食源性致病菌的快速分析提供思路。     

Potential of MALDI-TOF mass spectrometry as a rapid detection technique in plant pathology: identification of plant-associated microorganisms

Plant diseases caused by plant pathogens substantially reduce crop production every year, resulting in massive economic losses throughout the world. Accurate detection and identification of plant pathogens is fundamental to plant pathogen diagnostics and, thus, plant disease management. Diagnostics and disease-management strategies require techniques to enable simultaneous detection and quantification of a wide range of pathogenic and non-pathogenic microorganisms. Over the past decade, rapid development of matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for characterization of microorganisms has enabled substantially improved detection and identification of microorganisms. In the biological sciences, MALDI-TOF MS is used to analyze specific peptides or proteins directly desorbed from intact bacteria, fungal spores, nematodes, and other microorganisms. The ability to record biomarker ions, in a broad m/z range, which are unique to and representative of individual microorganisms, forms the basis of taxonomic identification of microorganisms by MALDI-TOF MS. Recent advances in mass spectrometry have initiated new research, i.e. analysis of more complex microbial communities. Such studies are just beginning but have great potential for elucidation not only of the interactions between microorganisms and their host plants but also those among different microbial taxa living in association with plants. There has been a recent effort by the mass spectrometry community to make data from large scale mass spectrometry experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MALDI-TOF MS as a universal technique for detection of plant pathogens and non-pathogens. The effects of experimental conditions are sufficiently understood, reproducible spectra can be obtained from computational database search, and microorganisms can be rapidly characterized by genus, species, or strain.     

Selection and analytical applications of aptamers binding microbial pathogens

DNA aptamers specifically recognizing microbial cells and viruses have a range of analytical and therapeutic applications. This article describes recent advances in the development of aptamers targeting specific pathogens (e.g., live bacteria, whole viral particles, and virally-infected mammalian cells). Specific aptamers against pathogens have been used as affinity reagents to develop sandwich assays, to label and to image cells, to bind with cells for flow-cytometry analysis, and to act as probes for development of whole-cell biosensors. Future applications of aptamers to pathogens will benefit from recent advances in improved selection and new aptamers containing modified nucleotides, particularly slow off-rate modified aptamers (SOMAmers).     

Microbial biosensors

A microbial biosensor is an analytical device that couples microorganisms with a transducer to enable rapid, accurate and sensitive detection of target analytes in fields as diverse as medicine, environmental monitoring, defense, food processing and safety. The earlier microbial biosensors used the respiratory and metabolic functions of the microorganisms to detect a substance that is either a substrate or an inhibitor of these processes. Recently, genetically engineered microorganisms based on fusing of the lux, gfp or lacZ gene reporters to an inducible gene promoter have been widely applied to assay toxicity and bioavailability. This paper reviews the recent trends in the development and application of microbial biosensors. Current advances and prospective future direction in developing microbial biosensor have also been discussed.     

Nucleic Acid Nanoprobes for Biosensor Development in Complex Matrices

Nucleic acid probes in living organisms play an essential role in therapeutics and diagnosis.Through the imaging and sensing of nucleic acid probes in complex biological matrices,a variety of diseases-related biological process,pathogenic process,or pharmacological responses to a therapeutic intervention have been discovered.However,a critical challenge of nucleic acid probes applied in complex matrices lies in enhancing the stability of nucleic acid probes,especially when it suffers from nuclease degradation and protein adsorption.In order to enhance the application of nucleic acid nanoprobes in complex matrices,great efforts have been devoted to improving the stability of probes operated in complex media,including construction of nucleic acid nanoprobes with nuclease resistance and protein adsorption resistance,sample pretreatment,anti-biofouling and signal correction.In this review,we aim to summarize recent advances in the stability of nucleic acid nanoprobes in complex matrices,including the methods of enhancing the stability of probes or signals,and the application of nucleic acid nanoprobes for disease diagnosis.     

多重聚合酶链反应-毛细管电泳-激光诱导荧光法检测三种食源性致病菌

建立了食品中常见致病菌大肠杆菌O157:H7的uidA基因、沙门菌的invA基因和志贺菌的ipaH基因的多重聚合酶链反应（PCR）产物的毛细管电泳快速检测方法。根据这3种致病菌的特异性基因序列设计多重PCR引物,优化PCR扩增反应体系,采用7.0 g/L 甲基纤维素为筛分介质,毛细管电泳-激光诱导荧光检测法同时检测了3种常见致病菌的PCR扩增产物。在优化的多重PCR反应和毛细管筛分电泳条件下,该方法可以同时检测沙门菌、志贺菌和大肠杆菌O157:H7基因的多重PCR扩增产物,22 min内即可完成3种常见致病菌的毛细管电泳检测。迁移时间的相对标准偏差为1.47%~2.07%。与凝胶电泳法比较,该法简便快速,灵敏度高,可用于多种致病菌脱氧核糖核酸的检测,为食品安全提供了一种可靠的快速检测方法。     

多响应曲面优化-毛细管电泳-激光诱导荧光快速检测食源性致病菌

建立了食品中常见致病菌: 沙门菌的invA基因、大肠杆菌O157∶H7的rfbO157基因、志贺菌的ipaH基因及副溶血性弧菌Vpara(16S-23S rDNA IGS)基因的多重PCR产物-毛细管电泳快速检测方法. 根据沙门菌、大肠杆菌O157∶H7、志贺菌及副溶血性弧菌的特异性基因保守序列设计出多重PCR引物, 优化PCR 扩增反应体系. 采用多响应曲面法优化毛细管电泳的分离条件, 以含有DNA荧光染料SYBR Green Ⅰ的1.0％甲基纤维素为筛分介质, 通过毛细管电泳-激光诱导荧光同时检测4种常见致病菌的PCR 扩增产物. 在优化的多重PCR反应体系和毛细管筛分电泳条件下, 此方法可以同时检测出沙门菌的invA基因、大肠杆菌O157∶H7的rfbO157基因、志贺菌的ipaH基因及副溶血性弧菌Vpara(16S-23S rDNA IGS)基因的多重PCR扩增产物, 25 min内即可完成检测. 迁移时间的日内相对标准偏差为0.92%~1.58%. 通过多响应曲面的优化, 有效改善了毛细管电泳对DNA分子的分离能力.     

Biocide Use in the Antimicrobial Era: A Review

Biocides are widely used in healthcare and industry to control infections and microbial contamination. Ineffectual disinfection of surfaces and inappropriate use of biocides can result in the survival of microorganisms such as bacteria and viruses on inanimate surfaces, often contributing to the transmission of infectious agents. Biocidal disinfectants employ varying modes of action to kill microorganisms, ranging from oxidization to solubilizing lipids. This review considers the main biocides used within healthcare and industry environments and highlights their modes of action, efficacy and relevance to disinfection of pathogenic bacteria. This information is vital for rational use and development of biocides in an era where microorganisms are becoming resistant to chemical antimicrobial agents.     

小儿尿路感染的微生物病原菌分布及解决对策

目的探讨小儿尿路感染的病原菌分布以及解决对策。方法选择90例小儿尿路感染患者为分析对象,分析其尿路感染病原微生物分布以及抗菌药物药敏情况。结果革兰阴性杆菌所占的比例达到了83.65%,其中大肠埃希菌占到71.26%。大肠埃希菌对头孢呱酮、头孢美蛙、亚胺培南、阿米卡星、氨曲南这些药物的敏感性较高。结论导致小儿尿路感染的主要病原微生物为大肠埃希菌,临床应依据病原菌分布以及抗菌药物药敏情况,合理选择和使用抗菌药物,有效控制感染,减少耐药菌株出现。     

毛细管电泳技术在疾病预防控制领域的应用、发展与挑战

自1989年出现商品化的仪器以来,毛细管电泳（CE）技术在多个应用领域都取得了长足的进步与发展,重复性和准确性方面也有很大提升。能力验证样品分析的满意结果也显示了CE具备法规要求的准确定量能力。在疾病预防控制领域（简称"疾控"）CE也展现出很多独具特色的应用,成为不可或缺的技术之一。在聚合酶链式反应产物分析、核酸序列测定、DNA变异和分型分析、食源性致病微生物分析及疫苗分析等工作中CE发挥了重要作用。应对突发疫情或公共卫生事件如食物中毒时,除了通过非靶标分析尽快锁定目标物外,还需要对大量样品做出快速而准确的分析,高通量和高灵敏的CE就十分适合解决这一问题。在公共卫生理化检验以确保食品、保健食品、特殊医学用途食品、化妆品和消毒产品等的安全中,CE也发挥了不可或缺的作用。作为一种使用较少危险化学品的环境友好方法,在需要按照标准或规范进行的疾控实验室常规检测中,CE仍受制于标准方法的缺失,未能发挥其应有的作用。但简单、快速、经济、耐用、高效的CE分离一旦与高灵敏通用检测器联用,必将更加从容地应对疾控领域中的各种挑战,发挥更大的作用。本文综述了2010~2019年CE在疾控领域的应用,分析了CE在疾控领域发展的机遇和挑战,对CE在疾控领域的发展前景进行了展望。     

A quick and easy method to identify bacteria by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry

Concerns with water quality have increased in recent years, in part due to the more frequent contamination of water by pathogens like E. coli and L. pneumophila. Current methods for the typing of bacteria in water samples are based on culture of samples on specific media. These techniques are time‐consuming, subject to the impact of interferents and do not totally meet all the requirements of prevention. There is a need for accurate and rapid identification of these microorganisms. This report deals with the detection of bacteria, more precisely of Legionella spp., and the development of an analytical strategy for a rapid and unambiguous identification of these pathogens in water from diverse origins. Therefore, a protein mass mapping using matrix‐assisted laser desorption/ionisation mass spectrometry (MALDI MS) of whole bacteria combined with a home‐made database of bacteria spectra is applied. A large variety of different bacteria and microorganisms is used to approach the actual composition of samples with numerous interferents. The objective is to propose a universal method for sampling preparation before MALDI MS analysis and optimised spectrometric conditions for reproducible intense peaks. Several experimental factors known to influence signal quality such as time and media of culture have been studied. The proposed method gives promising results for a sure differentiation of Legionella species and subspecies and a rapid identification of bacteria which are the most dangerous or difficult to eradicate. This method is easy to perform with an excellent reproducibility. The analytical protocol and the corresponding database were validated on samples from different origins (cooling tower, plumbing hot water). Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid detection of Staphylococcus aureus by a combination of monoclonal antibody-coated latex and capillary electrophoresis

The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S. aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S. aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S. aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0 x 10(5) colony forming unit/mL. We have also utilized this technology to identify S. aureus in a stool sample coming from a healthy volunteer spiked successfully with S. aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S. aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available.     

Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection

Foodborne illnesses caused by pathogenic bacteria represent a widespread and growing problem to public health, and there is an obvious need for rapid detection of food pathogens. Traditional culture-based techniques require tedious sample workup and are time-consuming. It is expected that new and more rapid methods can replace current techniques. To enable large scale screening procedures, new multiplex analytical formats are being developed, and these allow the detection and/or identification of more than one pathogen in a single analytical run, thus cutting assay times and costs. We review here recent advancements in the field of rapid multiplex analytical methods for foodborne pathogenic bacteria. A variety of strategies, such as multiplex polymerase chain reaction assays, microarray- or multichannel-based immunoassays, biosensors, and fingerprint-based approaches (such as mass spectrometry, electronic nose, or vibrational spectroscopic analysis of whole bacterial cells), have been explored. In addition, various technological solutions have been adopted to improve detectability and to eliminate interferences, although in most cases a brief pre-enrichment step is still required. This review also covers the progress, limitations and future challenges of these approaches and emphasizes the advantages of new separative techniques to selectively fractionate bacteria, thus increasing multiplexing capabilities and simplifying sample preparation procedures.

Surface plasmon resonance biosensors for detection of pathogenic microorganisms: strategies to secure food and environmental safety

This review describes the exploitation of exclusively optical surface plasmon resonance (SPR) biosensors for the direct and indirect detection of pathogenic microorganisms in food chains and the environment. Direct detection is, in most cases, facilitated by the use of defined monoclonal or polyclonal antibodies raised against (a part of) the target pathogenic microorganisms. The antibodies were immobilized to a solid phase of the sensor to capture the microbe from the sample. Alternatively, antibodies were used in an inhibition-like assay involving incubation with the target organism prior to analysis of nonbound antibodies. The free immunoglobins were screened on a sensor surface coated with either purified antigens or with Fc or Fab binding antibodies. Discussed examples of these approaches are the determination of Escherichia coli O1 57:H7, Salmonella spp., and Listeria monocytogenes. Another direct detection strategy involved SPR analysis of polymerase chain reaction products of Shiga toxin-2 genes reporting the presence of E. coli O157:H7 in human stool. Metabolic products have been exploited as biomarkers for the presence of a microbial agent, such as enterotoxin B and a virulence factor for the occurrence of Staphylococcus aureus and Streptococcus suis, respectively. Indirect detection, on the other hand, is performed by analysis of a humoral immune response of the infected animal or human. By immobilization of specific antigenic structures, infections with Herpes simplex and human immunodeficiency viruses, Salmonella and Treponema pallidum bacteria, and Schistosoma spp. parasites were revealed using human, avian, and porcine sera and avian eggs. Bound antibodies were easily isotyped using an SPR biosensor to reveal the infection history of the individual. Discussed studies show the recent recognition of the suitability of this type of instrument for (rapid) detection of health-threatening microbes to food and environmental microbial safety.     

聚集诱导发光分子在微生物检测和抗菌治疗中的应用

微生物检测和抗菌治疗与人类健康息息相关,快速检测和有效清除病原微生物对疾病治疗至关重要。传统的微生物检测方法如酶联免疫吸附检测（ELISA）、聚合酶链式反应（PCR）技术等,通常操作步骤复杂、耗时长且对仪器要求较高。另一方面,耐药性微生物的出现使得新型抗菌疗法的研发成为亟待解决的问题。聚集诱导发光分子（AIEgens）由于其出色的荧光和光敏性能,在微生物检测和抗菌治疗中表现出巨大的应用潜力。本文对AIEgens在微生物检测中的应用进行简要综述,总结了基于AIEgens的光动力治疗（photodynamic therapy, PDT）在细菌感染和多重耐药性细菌的清除中的应用研究,并对存在的不足以及未来的发展前景进行了讨论和展望。     

基于识别分子的磁分离技术在食源性致病菌分离中应用

食源性致病菌是一类对人类健康危害极大的致病菌,但由于其在食品基质中数量较少,通常需要在检测前对其进行富集。磁分离作为一种常用的前处理手段,在致病菌的富集、分离方面展示出极好的应用前景。常用于修饰在磁性材料上的识别分子有多种,本文主要综述了抗体、抗生素、核酸、凝集素等主要的几种识别分子在磁分离中的应用。     

适配体生物传感器在病原微生物检测中的应用

适配体是通过指数富集系统进化技术(SELEX)体外筛选得到的一类能够特异性地结合小分子物质、蛋白,甚至整个细胞的寡核苷酸序列.由于具有制备简便、易于修饰、稳定性好等特点,适配体已广泛应用于构建生物传感器,实现对病原微生物的识别和检测.本文在阐述适配体基本原理的基础之上,结合近年来病原微生物适配体研究领域的最新研究成果,综述以病原微生物为目标的适配体筛选技术的最新进展;列举目前已经筛选获得的病原微生物(原生生物、病毒、细菌)适配体;综述适配体生物传感器在病原微生物检测中的应用.并展望了适配体生物传感器在病原微生物检测领域的发展趋势.     

Rapid separation of microorganisms by quartz microchip capillary electrophoresis.

We developed and optimized a system coupling microchip capillary electrophoresis (MCE) and laser-induced fluorescence (LIF) detection for the analysis of microorganisms. The MCE-LIF system successfully separated pure cultures of lactic acid bacteria and Saccharomyces cerevisiae within 200 s. The results indicate that the MCE system can be conveniently used for the rapid and highly sensitive detection of microorganisms. Thus, MCE can provide a cheap and simple method for the on-line detection of microbial contamination.