The use of NASBA for the detection of microbial pathogens in food and environmental samples

The isothermal amplification method nucleic acid sequence-based amplification (NASBA), which amplifies RNA, has been reported as useful for the detection of microbial pathogens in food and environmental samples. Methods have been published for Campylobacter spp., Listeria monocytogenes and Salmonella enterica ser. Enteritidis in various foods and for Cryptosporidium parvum in water. Both 16S rRNA and various mRNAs have been used as target molecules for detection; the latter may have advantages in allowing specific detection of viable cells. Most of the methods to detect pathogens in foods have employed enrichment in nutrient medium prior to NASBA, as this can ensure sensitivity of detection and encourage the detection of only viable target cells. Although a relatively recent method, NASBA has the potential for adoption as a diagnostic tool for environmental pathogens.     

基于核酸侵入反应的基因突变检测方法研究进展

肿瘤靶向药物治疗需要检测特异性的基因突变。尽管已开发出多种基于模板扩增的基因突变检测方法,但由于存在扩增产物交叉污 染的风险,其应用受到极大限制。基于信号扩增的核酸侵入反应,由于不存在扩增产物污染的风险,并且具有良好的单碱基识别特异性, 非常适用于基因突变的检测。因此,一系列基于核酸侵入反应的基因突变检测方法应运而生。综述若干基于核酸侵入反应的基因突变检测 方法原理与应用研究。     

Microarrays for pathogen detection and analysis

DNA microarrays have emerged as a viable platform for detection of pathogenic organisms in clinical and environmental samples. These microbial detection arrays occupy a middle ground between low cost, narrowly focused assays such as multiplex PCR and more expensive, broad-spectrum technologies like high-throughput sequencing. While pathogen detection arrays have been used primarily in a research context, several groups are aggressively working to develop arrays for clinical diagnostics, food safety testing, environmental monitoring and biodefense. Statistical algorithms that can analyze data from microbial detection arrays and provide easily interpretable results are absolutely required in order for these efforts to succeed. In this article, we will review the most promising array designs and analysis algorithms that have been developed to date, comparing their strengths and weaknesses for pathogen detection and discovery.     

Using hierarchical models to compare the sensitivity of metabarcoding and qPCR for eDNA detection

Environmental DNA (eDNA) sampling—the detection of intra- or extra-cellular DNA in environmental samples—is a rapid and sensitive survey method for detecting aquatic species. Single-species detection methods (typically based on PCR or LAMP) have been shown to be more sensitive for detecting target species than multi-species detection methods, such as metabarcoding. However, previous studies have generally only compared these two eDNA detection approaches for a single target species and have used different methodological and statistical approaches. Here we present a comparison of single- and multi-species eDNA detection methods, drawing on two published case studies (one fish, one amphibian) and two new extensive datasets on a freshwater mammal (the platypus). To ensure consistent conclusions regarding the sensitivity of each eDNA method, we use the same hierarchical site occupancy-detection model for each dataset, incorporating uncertainty at the site, water sample, and technical replicate level. Overall, qPCR achieved higher detection probabilities than metabarcoding across species and datasets. However, differences in sensitivity between detection methods varied depending on methodological decisions concerning what constitutes a true positive detection (i.e., qPCR and metabarcoding thresholds). The decision as to which eDNA detection method to use should always be influenced by the study aims, but our results suggest that single-species detection methods based on qPCR may be preferable when the aim is to achieve a high detection probability for target species.     

用于新型冠状病毒检测的核酸等温扩增技术研究进展

核酸检测作为新型冠状病毒肺炎(COVID-19)筛查诊断和病情监测的主要手段,在疫情防控中发挥了重要作用。虽然实时荧光定量PCR被认为是新型冠状病毒(SARS-CoV-2)核酸检测的金标准,但其依赖荧光定量PCR仪且扩增检测时间较长,难以实现现场快速检测。因此许多基于核酸等温扩增的SARS-CoV-2检测方法相继诞生。等温扩增对仪器温控要求不高,通过与微流控芯片和可视化检测技术结合,可进一步简化操作、降低成本,为SARS-CoV-2现场快速筛查提供有力的技术支撑。本文围绕已报道的SARS-CoV-2等温扩增检测方法原理、检测性能及优缺点进行探讨,为进一步发展SARS-CoV-2现场快速检测平台提供参考。     

Maltose-binding protein: a versatile platform for prototyping biosensing

The bacterial periplasmic-binding protein (PBP) superfamily members, in particular the maltose-binding protein, have been used extensively to prototype a variety of biosensing platforms. Although quite diverse at the primary sequence level, this protein superfamily retains the same basic two-domain structure, and upon binding a recognized ligand almost all PBPs undergo a conformational change to a closed structure. This process forms the basis for most, but not all, PBP-based biosensor signal transduction. Many direct detection or reagentless sensing modalities have been utilized with maltose-binding protein for both in vitro and in vivo detection of target compounds. Signal transduction modalities developed to date include direct fluorescence, electrochemical detection, fluorescence resonance energy transfer (FRET)-based detection, surface-tethered FRET sensing, hybrid quantum dot FRET sensing, and enzymatic detection, each of which have different benefits, potential applications and limitations.     

Analysis of mutagenic imidazo[4,5-f]quinolines and -quinoxalines (IQ compounds). Comparison of electrochemical and ultraviolet detection

Two synthetic imidazoquinolin-2-amines (IQ and MeIQ) and two imidazoquinoxalin-2-amines (MeIQx and 4,8-DiMeIQx), all known potent mutagens, have been separated by reversed-phase HPLC and detected by two methods - UV detection and electrochemical (EC) detection. The limits of detection were found to be 2.5 pmoles for UV detection and 0.5-1.5 pmoles for electrochemical detection.     

Advances in molecular detection of Aspergillus: an update

Filamentous cosmopolitan fungi of the genus Aspergillus can be harmful in two ways, directly they can be opportunistic pathogens causing aspergillosis and indirectly due to aflatoxin production on food products which can lead to aflatoxicosis. Therefore, a number of methods have been proposed so far for detection of the fungi with lowest possible concentration at the earliest. Molecular methods such as PCR and/or in combination with certain techniques have been found to be useful for Aspergillus detection. We discuss here various technologies that have emerged in recent years and can possibly be used for the molecular detection of Aspergillus in an efficient way. These methods like RSIC, C-probe, and inversion probe with pyrosequencing or direct ss/dsDNA detection have been used for the identification of fungal or bacterial pathogens and thus formulate a ‘gold standard’ for Aspergillus detection.     

Application of electronic estrus detection technologies to reproductive management of cattle.

Artificial insemination and embryo transfer programs are dependent on efficient and accurate detection of estrus. Visual observation is accurate at detecting animals in estrus, but efficiency ranges from approximately 50 to 70%. Electronic technologies have been developed in attempts to improve estrus detection efficiency. Commercially available electronic devices for estrus detection are based on changes in physical activity (pedometers), changes in electrical resistance of reproductive tract secretions (intravaginal resistance probes) or mounting activity (mount detectors). All of the commercially available electronic estrus detection devices can improve the efficiency of estrus detection in cattle. Pedometers are most applicable to lactating dairy cattle and have greater accuracy and efficiency when combined with visual observation. Intravaginal resistance measurement is perhaps the least practical method of estrus detection because of labor and animal handling requirements. Individual resistance measurement may have practical application for confirming other inconclusive signs of estrus. Mount monitors have the broadest application to beef and dairy cattle. HeatWatch, the only real-time radiotelometric system available, requires the least labor and animal handling and provides data on the time and duration of each mount. The less expensive stand-alone mount monitors also provide the necessary information for optimum timing of insemination and embryo transfer, but are more labor intensive.     

综述与专论: 河鲀毒素的检测技术与应用

河鲀毒素（tetrodotoxin， TTX）是毒性极强的小分子生物碱类毒素，包括中国在内的亚洲沿海国家因误食TTX污染食品而中毒的事件时有发生，其发病迅速且无特效解毒剂，对环境安全、食品安全与社会安全造成极大的威胁。通过检测食品与环境中的TTX含量可以实现TTX的风险预警，可有效防范TTX中毒事件的发生。本文梳理了4类TTX的检测技术，分析比较了传统的生物检测法、化学检测法、免疫检测法之间的优势、不足与实际应用进展，介绍了基于适配体技术的新型检测技术的兴起、发展与广阔的应用前景，对生物安全领域中TTX风险的管理与控制有现实意义。     

食源性病毒核酸恒温检测技术研究进展

食源性病毒已成为全球引发食品安全事件的重要病原,对新型检测技术的不断发展提出了严峻的挑战.早期PCR技术在病原检测领域中的应用,推动了对食源性病毒的全面认识.近年来核酸恒温检测技术发展迅速,包括环介导等温扩增技术、重组酶聚合酶扩增技术、核酸序列依赖性扩增技术、链置换扩增技术、滚环扩增技术等,在抗复杂基质干扰、装备要求低...     

食品中单核增生李斯特氏菌检测研究进展

单核增生李斯特氏菌和李斯特菌病的危害近年来引起世界各国食品和卫生部门的广泛关注.关于如何找到一种快速、敏感、准确、合理的检验方法,是当今各国食品卫生部门亟待解决的重要研究课题.对该菌的传统分离方法、免疫学检测方法、核酸检测等方法的最新进展进行了综述,为进行该菌的准确、快速检测奠定了基础.     

Biomolecular computation with molecular beacons for quantitative analysis of target nucleic acids

Molecular beacons are efficient and useful tools for quantitative detection of specific target nucleic acids. Thanks to their simple protocol, molecular beacons have great potential as substrates for biomolecular computing. Here we present a molecular beacon-based biomolecular computing method for quantitative detection and analysis of target nucleic acids. Whereas the conventional quantitative assays using fluorescent dyes have been designed for single target detection or multiplexed detection, the proposed method enables us not only to detect multiple targets but also to compute their quantitative information by weighted-sum of the targets. The detection and computation are performed on a molecular level simultaneously, and the outputs are detected as fluorescence signals. Experimental results show the feasibility and effectiveness of our weighted detection and linear combination method using molecular beacons. Our method can serve as a primitive operation of molecular pattern analysis, and we demonstrate successful binary classifications of molecular patterns made of synthetic oligonucleotide DNA molecules.     

荧光单分子检测技术在生命科学中的应用

荧光单分子检测技术是用荧光标记来显示和追踪单个分子的构象变化、动力学,单分子之间的相互作用以及单分子操纵的研究。过去对于生命科学分子机制的研究,都是对分子群体进行研究,然后平均化来进行单分子估测。因此,单个分子的动态性和独立性也被平均化掉而无法表现出来。荧光单分子检测技术真正实现了对单个分子的实时观测,将过去被平均化并隐藏在群体测量中不能获得的信息显示出来。近几年来,荧光单分子检测技术的飞速发展,为生命科学的发展,开辟了全新的研究领域。现就荧光单分子检测技术在研究动力蛋白、DNA转录、酶反应、蛋白质动态性和细胞信号转导方面的应用进展作一综述。     

Clinical applications of molecular biology for infectious diseases

Molecular biological methods for the detection and characterisation of microorganisms have revolutionised diagnostic microbiology and are now part of routine specimen processing. Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods. In addition to detection of fastidious microorganisms, more rapid detection by molecular methods is now possible for pathogens of public health importance. Molecular methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterisation by genotyping. Treatment of certain microorganisms has been improved by viral resistance detection and viral load testing for the monitoring of responses to antiviral therapies. With the advent of multiplex PCR, real-time PCR and improvements in efficiency through automation, the costs of molecular methods are decreasing such that the role of molecular methods will further increase. This review will focus on the clinical utility of molecular methods performed in the clinical microbiology laboratory, illustrated with the many examples of how they have changed laboratory diagnosis and therefore the management of infectious diseases.     

脑循环功能检测技术近三十年进展

脑循环功能的异常改变往往早于形态学和影像学改变,这对于脑血管的早期检测以及治疗过程中疗效的评价有显著意义。国内外脑循环功能检测技术的发展已有30年左右的历史,本文综述了脑循环动力学参数的生理背景及脑循环功能检测技术近年来的发展。     

An overview of transducers as platform for the rapid detection of foodborne pathogens

The driving advent of portable, integrated biosensing ways for pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques. The miniaturization and automation of integrated detection systems present a significant advantage for rapid, portable detection of foodborne microbes. In this review, we have highlighted current developments and directions in foodborne pathogen detection systems. Recent progress in the biosensor protocols toward the detection of specific microbes has been elaborated in detail. It also includes strategies and challenges for the implementation of a portable platform toward rapid foodborne sensing systems.     

Bacillus spore inactivation methods affect detection assays

Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Assays for detection in the laboratory often employ inactivated preparations of spores or nonpathogenic simulants. This study uses several common biodetection platforms to detect B. anthracis spores that have been inactivated by two methods and compares those data to detection of spores that have not been inactivated. The data demonstrate that inactivation methods can affect the sensitivity of nucleic acid- and antibody-based assays for the detection of B. anthracis spores. These effects should be taken into consideration when comparing laboratory results to data collected and assayed during field deployment.     

Cancer detection centers; the experience to date in California

Cancer "detection centers" (that is, centers for the examination of presumably well or asymptomatic persons) have been tried out in four different California communities during the last three years. In all instances-as in most other such centers throughout the United States-they have not been successful in restricting examination to well persons.The detection centers in California may therefore be described more accurately as "cancer examination and detection clinics."Three of the four centers have been closed owing to the small yield of cancer cases discovered, plus the fact that the cost of operation exceeded the total available funds of the local branch of the Cancer Society. In addition, it was extremely difficult to obtain and maintain competence on the part of the professional staff in such centers.A more practical approach to the problem of earlier tumor detection would appear to be emphasis on making "every physician's office a detection center," and stressing the annual examination of persons over 40 years of age for tumors in the five common accessible sites. These are the tumors most readily curable today.     

Protein-protein interaction studies on protein arrays: Effect of detection strategies on signal-to-background ratios

Protein arrays hold great promise for proteome-scale analysis of protein-protein interaction networks, but the technical challenges have hindered their adoption by proteomics researchers. The crucial issue of design and fabrication of protein arrays have been addressed in several studies, but the detection strategies used for identifying protein-protein interactions have received little attention. In this study, we evaluated six different detection strategies to identify four different protein-protein interaction pairs. We discuss each detection approach in terms of signal-to-background (S/B) ratio, ease of use, and adaptability to high-throughput format. Protein arrays for this study were made by expressing both the bait proteins (proteins captured at the surface) and prey proteins (probes) in cell-free rabbit reticulocyte lysate (RRL) systems. Bait proteins were expressed as HaloTag fusions that allow covalent capture on a HaloTag ligand-coated glass without any prior protein purification step. Prey proteins were expressed and modified with either tags (protein or peptides) or labels (fluorescent or radiometric) for detection. This simple method for creating protein arrays in combination with our analyses of several detection strategies should increase the usefulness of protein array technologies.