An overview of foodborne pathogen detection: In the perspective of biosensors

Food safety is a global health goal and the foodborne diseases take a major crisis on health. Therefore, detection of microbial pathogens in food is the solution to the prevention and recognition of problems related to health and safety. For this reason, a comprehensive literature survey has been carried out aiming to give an overview in the field of foodborne pathogen detection. Conventional and standard bacterial detection methods such as culture and colony counting methods, immunology-based methods and polymerase chain reaction based methods, may take up to several hours or even a few days to yield an answer. Obviously this is inadequate, and recently many researchers are focusing towards the progress of rapid methods. Although new technologies like biosensors show potential approaches, further research and development is essential before biosensors become a real and reliable choice. New bio-molecular techniques for food pathogen detection are being developed to improve the biosensor characteristics such as sensitivity and selectivity, also which is rapid, reliable, effective and suitable for in situ analysis. This paper not only offers an overview in the area of microbial pathogen detection but it also describes the conventional methods, analytical techniques and recent developments in food pathogen detection, identification and quantification, with an emphasis on biosensors.     

Market analysis of biosensors for food safety

This paper is presented as an overview of the pathogen detection industry. The review includes pathogen detection markets and their prospects for the future. Potential markets include the medical, military, food, and environmental industries. Those industries combined have a market size of $563 million for pathogen detecting biosensors and are expected to grow at a compounded annual growth rate of 4.5%. The food market is further segmented into different food product industries. The overall food-pathogen testing market is expected to grow to $192 million and 34 million tests by 2005. The trend in pathogen testing emphasizes the need to commercialize biosensors for the food safety industry as legislation creates new standards for microbial monitoring. With quicker detection time and reusable features, biosensors will be important to those interested in real time diagnostics of disease causing pathogens. As the world becomes more concerned with safe food and water supply, the demand for rapid detecting biosensors will only increase.     

Electrical/electrochemical impedance for rapid detection of foodborne pathogenic bacteria

The realization of rapid, sensitive, and specific methods to detect foodborne pathogenic bacteria is central to implementing effective practice to ensure food safety and security. As a principle of transduction, the impedance technique has been applied in the field of microbiology as a means to detect and/or quantify foodborne pathogenic bacteria. The integration of impedance with biological recognition technology for detection of bacteria has led to the development of impedance biosensors that are finding wide-spread use in the recent years. This paper reviews the progress and applications of impedance microbiology for foodborne pathogenic bacteria detection, particularly the new aspects that have been added to this subject in the past few years, including the use of interdigitated microelectrodes, the development of chip-based impedance microbiology, and the use of equivalent circuits for analysis of the impedance systems. This paper also reviews the significant developments of impedance biosensors for bacteria detection in the past 5 years, focusing on microfabricated microelectrodes-based and microfluidic-based Faradaic electrochemical impedance biosensors, non-Faradaic impedance biosensors, and the integration of impedance biosensors with other techniques such as dielectrophoresis and electropermeabilization.     

生物传感器在食源性致病菌检测中应用的研究进展

食源性致病菌作为引起食源性疾病的主要因素,受到人们的高度重视,发展简便、快速、高灵敏度和低成本的食源性致病菌检测方法对降低食源性疾病发病率具有重要意义。生物传感器技术是一种由多学科交叉渗透发展形成的全新微量分析技术,具有灵敏度高、分析速度快等特点,被广泛应用于食源性致病菌的检测。文中介绍了生物传感器的基本原理,综述了常见的生物传感器在食源性致病菌检测中的应用,并对其发展趋势进行了展望。     

植物病原细菌检测和细菌病害诊断方法

植物病害诊断是通过病菌鉴定和致病性测定等步骤实现的。在过去的10年里用于细菌鉴定的基因组技术的快速发展极大地简化和促进了病菌的检测和鉴定,但是DNA分子检测方法不能完全取代传统的培养检验和表型检验方法。文中介绍了未显示病害症状的植物或植物产品中已知细菌的免疫检测和基因组DNA检测方法,以及细菌病害诊断鉴定的新方法。     

Rapid PCR confirmation of E. coli O157:H7 after evanescent wave fiber optic biosensor detection

Escherichia coli O157:H7 is an enteric pathogen of public health importance, which is monitored by several government agencies. Many rapid detection tests have been developed to identify foodstuff and water supplies contaminated by E. coli O157:H7. However, these methods can be time consuming (24-48 h) due to the need to culture the bacteria to confirm detection results. Fiber optic biosensors can rapidly detect pathogens from complex matrices, yet confirmation tests can take up to 10h to complete. In addition, fiber optic biosensors can also be used to reduce the impact of PCR inhibitors present in complex matrices such as food and water. This paper presents methodologies to reduce the time necessary for confirmation from 10 to about 2 h, by direct PCR of bacteria from the fiber optic waveguides without the need for culture or enrichment steps.     

基于CRISPR/Cas生物传感原理的病原菌检测技术研究进展*

病原菌的快速准确检测是实现疫情高效防控、疾病精准治疗、污染环境及时处置的关键。而现有的病原菌现场快速检测技术,主要以定性分析为主,假阳性/假阴性受到诟病,检测准确性仍有待提升,亟待发展基于新原理、新方法的病原菌快速检测技术。基于CRISPR(clustered regularly interspaced short palindromic repeats)的生物传感技术因具有高灵活性(对不同的基因靶点只需改变crRNA序列)、高特异性(单碱基分辨)、高灵敏(优于10^-18 mol/L浓度)、可编程、可模块化、低成本、可在各种体外介质中高效稳定运行等独特优势,打破了传统分子诊断与检测技术的局限性,正在成为下一代病原菌检测技术的引领者。在该技术中,Cas效应蛋白被用作高特异性的序列识别元件,结合不同的生物传感机制,即可用于病原菌的高特异性快速灵敏检测。在总结CRISPR/Cas生物传感技术原理的基础上,综述了用于病原菌检测的CRISPR/Cas12和CRISPR/Cas13生物传感技术研究进展。通过阐述CRISPR/Cas生物传感技术在实际应用中面临的挑战,展望其未来的发展前景。     

常见生物传感芯片及其应用进展

生物传感芯片是一类综合了生物芯片和生物传感器的优点的新型生物芯片,在保持传统生物芯片的高通量、可寻址、并行处理等特点的基础上,与生物传感器技术相结合,进一步提高了芯片检测的灵敏度和特异性。常见的生物传感芯片主要有光纤传感芯片、表面等离子体共振传感芯片、热生物传感芯片、压电晶体传感芯片等,可用于各种生物大分子,如蛋白质、核酸等的检测,金属离子的测定,病原体的检测,药物筛选等。     

Developing nucleic acid-based electrical detection systems

Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detection are discussed.     

Current trends in endotoxin detection and analysis of endotoxin–protein interactions

Endotoxin is a type of pyrogen that can be found in Gram-negative bacteria. Endotoxin can form a stable interaction with other biomolecules thus making its removal difficult especially during the production of biopharmaceutical drugs. The prevention of endotoxins from contaminating biopharmaceutical products is paramount as endotoxin contamination, even in small quantities, can result in fever, inflammation, sepsis, tissue damage and even lead to death. Highly sensitive and accurate detection of endotoxins are keys in the development of biopharmaceutical products derived from Gram-negative bacteria. It will facilitate the study of the intermolecular interaction of an endotoxin with other biomolecules, hence the selection of appropriate endotoxin removal strategies. Currently, most researchers rely on the conventional LAL-based endotoxin detection method. However, new methods have been and are being developed to overcome the problems associated with the LAL-based method. This review paper highlights the current research trends in endotoxin detection from conventional methods to newly developed biosensors. Additionally, it also provides an overview of the use of electron microscopy, dynamic light scattering (DLS), fluorescence resonance energy transfer (FRET) and docking programs in the endotoxin–protein analysis.     

Exposing culprit organic pollutants: a review

There is a continuing need for monitoring the health of the environment due to the presence of pollutants. Here, we review the development and attributes of biosensors by which bacteria have been genetically modified to express the luminescence genes, i.e. to glow, in a quantified manner, in response to pollutants. We have concentrated on the detection of organic hydrocarbon pollutants and discussed the molecular mechanisms by which some of these chemicals act as effector molecules on the respective regulatory systems. The future of environmental biosensors is predictably bright. As more knowledge is gathered on the sensing regulatory component, the possibility of developing targeted or pollutant-specific biosensors is promising. Moreover, the repertoire of biosensors for culprit organic pollutants is expected to be enlarged through advances in genomics technology and identification of new sensory or receptor molecules. The need for pollutant detection at concentrations in the parts per trillion range or biosensors configured in a nanoscale is anticipated.     

Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula

Enterobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in neonates and children. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, we developed two real time PCR assays using TaqMan and SYBR Green to identify the pathogen after selective enrichment in mLST and BHI. The accuracy of two detections was tested by 35 strains of E. sakazakii and 88 non-E. sakazakii bacterial strains. The results showed that all of these E. sakazakii strains were positive reaction to the detections and all of the non-E. sakazakii strains were negative. The newly developed assays enable us to detect 1.1 CFU/100 g infant formula. And both of the assays can be accomplished within 2 business days. Compared to the traditional detection, the real time PCR procedures are quicker and simpler. In this study, we also developed a new method to design the primers, which can support multiple real time PCR with one pair of primers in SYBR Green detection. The detection methods are more sensitive and effective based on Two-Tm-Value of PCR.     

Simplified detection of food-borne pathogens: An in situ high affinity capture and staining concept

Food industries need simple, rapid and cost-effective solutions for pathogen detection in food and environmental samples. In this paper, we describe a simple but novel detection concept combining an affinity capture surface and intracellular metabolic marker to visualize the bacterial presence on the affinity surface. The surface of a Solid Phase Support (SPS) is functionalized with specific phage tail proteins targeted to the bacterial pathogen of interest. The SPS is placed directly into the primary food enrichment bag after stomaching. Following incubation, the captured bacteria are visually detected in situ as a result of the bacterial reduction of the colorless soluble substrate triphenyltetrazolium chloride (TTC) (present in the primary culture medium) to an intracellular red insoluble formazan product. Detection on the SPS is observed as an intense red color after 22 to 40 hours of enrichment. This is not impaired by the presence of food particles and the natural background microflora. The in situ method significantly simplifies pathogen detection by eliminating any post-enrichment intervention that is necessary in the traditional methods of analysis. We have demonstrated the application of this new approach for the detection of Escherichia coli O157: H7, Listeria spp. and Salmonella spp. in artificially contaminated food samples.     

From DNA biosensors to gene chips

Wide-scale DNA testing requires the development of small, fast and easy-to-use devices. This article describes the preparation, operation and applications of biosensors and gene chips, which provide fast, sensitive and selective detection of DNA hybridization. Various new strategies for DNA biosensors and gene chips are examined, along with recent trends and future directions. The integration of hybridization detection schemes with the sample preparation process in a ‘Lab-on-a-Chip’ format is also covered. While the use of DNA biosensors and gene chips is at an early stage, such devices are expected to have an enormous effect on future DNA diagnostics.     

核酸适配体光学生物传感器在卡那霉素检测中的研究进展

卡那霉素是一种氨基糖苷类抗生素,由于其效果好、价格低等优点,是我国常用兽药之一。但如果剂量过大就会大量残留在动物体内,进而通过食物链富集进入人体,引发耳毒性、肾毒性等毒副作用,严重时会导致人死亡,因此对其含量的检测十分重要。近几年,大量基于核酸适配体检测卡那霉素的光学方法被开发,以满足人们对其检测的需求。首先明确了核酸适配体与光学生物传感器等相关概念;再根据其反应机制不同,对光学方法介导的生物传感器进行了分类综述,阐述了各类传感器的基本原理及其检测范围;最后对这几类传感器的优缺点和发展前景进行了总结和展望,对这些方法的进一步探索将为食品安全检测提供新的技术支撑。     

Biosensor technologies for detecting microbiological foodborne hazards

The convergence of molecular biology and miniaturized instrumentation has accelerated development of biosensors with the specifications necessary to support pathogen reduction and quality programs in the food supply. Advances in optoelectronics, thin layer deposition, and microfabrication have provided many options for achieving microbiological detection goals. Some promising technologies are reviewed.     

Biosensors and bioelectrochemistry

This review describes recent developments in the field of biosensors and bioelectrochemistry. Nanoparticles have been used to improve sensor performance and to develop biosensors based on new detection principles. Their use has extended into all areas of biosensor and bioelectrochemistry research. Other active areas of biosensor development include DNA sensing, immunosensing, direct electron transfer between an electrode and a redox protein or enzyme, and in vivo sensors.     

The challenge to quantify Listeria monocytogenes--a model leading to new aspects in molecular biological food pathogen detection

In this work, we discuss the latest insights concerning advantages and disadvantages and the nature of microbiological and molecular methods for quantitative food pathogen detection. The assessment of molecular methods must be brought on a basis that considers the nature of molecular methods and their underlying mechanism. A potential approach to setting up the development, validation and structure of an analytical chain is presented based on quantitative real-time PCR (qPCR). This is analysed exemplary on the basis of recent work using the model organism Listeria monocytogenes. Several prerequisites for successful quantitative detection of this pathogen will be discussed. In particular, sample preparation, controls for all methodical steps and the validation of the core assay qPCR are addressed, which constitute the basis for a reliable analytical detection chain for molecular biological pathogen detection from food. Microbiological methods are analysed based on growth of the single cell, which is the fundament of these traditional methods.     

Making bio-sense of toxicity: new developments in whole-cell biosensors

Bacterial whole-cell biosensors are very useful for toxicity measurements of various samples. Semi-specific biosensors, containing fusions of stress-regulated promoters and reporter genes, have several advantages over the traditional, general biosensors that are based on constitutively expressed reporter genes. Furthermore, semi-specific biosensors are constantly being refined to lower their sensitivity and, in combination, are able to detect a wide range of toxic agents. However, the requirement for a positive response of these biosensors to toxicants can result in false-negative responses. The application of in situ inoculation and single-cell detection, combined with the introduction of new reporter genes and refined detection equipment, could lead to the extensive use of semi-specific, stress-responsive biosensors for toxicity estimations in the future.     

Measurement of purine release with microelectrode biosensors

Purinergic signalling departs from traditional paradigms of neurotransmission in the variety of release mechanisms and routes of production of extracellular ATP and adenosine. Direct real-time measurements of these purinergic agents have been of great value in understanding the functional roles of this signalling system in a number of diverse contexts. Here, we review the methods for measuring purine release, introduce the concept of microelectrode biosensors for ATP and adenosine and explain how these have been used to provide new mechanistic insight in respiratory chemoreception, synaptic physiology, eye development and purine salvage. We finish by considering the association of purine release with pathological conditions and examine the possibilities that biosensors for purines may one day be a standard part of the clinical diagnostic tool chest.