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...     

Research progress in the detection of common foodborne hazardous substances based on functional nucleic acids biosensors

With the further improvement of food safety requirements, the development of fast, highly sensitive, and portable methods for the determination of foodborne hazardous substances has become a new trend in the food industry. In recent years, biosensors and platforms based on functional nucleic acids, along with a range of signal amplification devices and methods, have been established to enable rapid and sensitive determination of specific substances in samples, opening up a new avenue of analysis and detection. In this paper, functional nucleic acid types including aptamers, deoxyribozymes, and G-quadruplexes which are commonly used in the detection of food source pollutants are introduced. Signal amplification elements include quantum dots, noble metal nanoparticles, magnetic nanoparticles, DNA walkers, and DNA logic gates. Signal amplification technologies including nucleic acid isothermal amplification, hybridization chain reaction, catalytic hairpin assembly, biological barcodes, and microfluidic system are combined with functional nucleic acids sensors and applied to the detection of many foodborne hazardous substances, such as foodborne pathogens, mycotoxins, residual antibiotics, residual pesticides, industrial pollutants, heavy metals, and allergens. Finally, the potential opportunities and broad prospects of functional nucleic acids biosensors in the field of food analysis are discussed.     

Molecular Detection, Quantification, and Diversity Evaluation of Microalgae

This study reviews the available molecular methods and new high-throughput technologies for their practical use in the molecular detection, quantification, and diversity assessment of microalgae. Molecular methods applied to other groups of organisms can be adopted for microalgal studies because they generally detect universal biomolecules, such as nucleic acids or proteins. These methods are primarily related to species detection and discrimination among various microalgae. Among current molecular methods, some molecular tools are highly valuable for small-scale detection [e.g., single-cell polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), and biosensors], whereas others are more useful for large-scale, high-throughput detection [e.g., terminal restriction length polymorphism, isothermal nucleic acid sequence-based amplification, loop-mediated isothermal amplification, microarray, and next generation sequencing (NGS) techniques]. Each molecular technique has its own strengths in detecting microalgae, but they may sometimes have limitations in terms of detection of other organisms. Among current technologies, qPCR may be considered the best method for molecular quantification of microalgae. Metagenomic microalgal diversity can easily be achieved by 454 pyrosequencing rather than by the clone library method. Current NGS, third and fourth generation technologies pave the way for the high-throughput detection and quantification of microalgal diversity, and have significant potential for future use in field monitoring.     

Alternative Molecular Tests for Virological Diagnosis

Several nucleic acid amplification techniques (NAATs), particularly PCR and real-time PCR, are currently used in the routine clinical laboratories. Such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. However, conventional PCR methods have several intrinsic disadvantages such as the requirement for temperature cycling apparatus, and sophisticated and costly analytical equipments. Therefore, amplification at a constant temperature is an attractive alternative method to avoid these requirements. A new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction and easy detection. The main isothermal methods reviewed here include loop-mediated isothermal amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. In this review, design criteria, potential of amplification, and application of these alternative molecular tests will be discussed and compared to conventional NAATs.     

硅对PCR过程的抑制作用研究

探讨不同氧化程度的硅材料对PCR扩增的抑制作用及其机理。将不同氧化程度的硅纳米颗粒加入PCR反应液中,使其与Taq酶、模板等充分接触,通过离心将硅纳米颗粒沉降在管壁上,取出上清或保留硅纳米颗粒上机扩增,扩增产物采用凝胶电泳法检测。结果表明,随着硅材料表面面积与PCR反应液体积之比的增大,核酸扩增效率将明显下降,并且在所研究的范围内,氧化程度高的硅材料对PCR过程抑制作用更强;通过对抑制作用机理进行初步的实验研究,表明硅材料对PCR反应液中的Taq酶的吸附是导致抑制现象产生的主要原因,而对模板的吸附影响较小;并且,反应管内是否保留硅材料对核酸扩增影响较小,硅材料没有明显的直接化学抑制作用。     

Feasibility of methods based on nucleic acid amplification techniques to fulfil the requirements for microbiological analysis of water quality

Molecular methods based on nucleic acid recognition and amplification are valuable tools to complement and support water management decisions. At present, these decisions are mostly supported by the principle of end‐point monitoring for indicators and a small number of selected measured by traditional methods. Nucleic acid methods show enormous potential for identifying isolates from conventional culture methods, providing data on cultivable and noncultivable micro‐organisms, informing on the presence of pathogens in waters, determining the causes of waterborne outbreaks, and, in some cases, detecting emerging pathogens. However, some features of water microbiology affect the performance of nucleic acid‐based molecular techniques and thus challenge their suitability for routine water quality control. These features include the variable composition of target water samples, the generally low numbers of target micro‐organisms, the variable water quality required for different uses and the physiological status or condition of such micro‐organisms. The standardization of these molecular techniques is also an important challenge for its routine use in terms of accuracy (trueness and precision) and robustness (reproducibility and reliability during normal usage). Most of national and international water regulations recommend the application of standard methods, and any new technique must be validated respect to established methods and procedures. Moreover, molecular methods show a high cost‐effectiveness value that limits its practicability on some microbial water analyses. However, new molecular techniques could contribute with new information or at least to supplement the limitation of traditional culture‐based methods. Undoubtedly, challenges for these nucleic acid‐based methods need to be identified and solved to improve their feasibility for routine microbial water monitoring.     

基于CRISPR/Cas系统的核酸生物传感器

近年来,CRISPR/Cas系统已经成为转录调控和基因组编辑的重要工具。除了在基因编辑领域的贡献,CRISPR/Cas系统独特的靶核酸顺式切割和非特异性单链核酸反式切割能力,在开发核酸检测的新型生物传感器方面展现出巨大潜力。构建基于CRISPR/Cas系统高灵敏度生物传感器的关键通常依赖其与不同信号扩增策略,诸如核酸扩增技术或特定信号转导方法的结合。基于此,本文旨在通过介绍不同类型的CRISPR/Cas系统,全面概述基于该系统的核酸检测生物传感器的研究进展,并重点对结合核酸扩增技术（PCR、LAMP、RCA、RPA和EXPAR）、灵敏的信号转导方法（电化学和表面增强拉曼光谱）和特殊结构设计生物传感的三大类型信号放大策略的CRISPR/Cas生物传感器进行总结和评论。最后,本文对目前的挑战以及未来的前景进行展望。     

Impact of molecular biology on the detection of foodborne pathogens

Molecular biological methods that use antibodies and nucleic acids to detect specific foodborne bacterial pathogens were scarcely known a decade and a half ago. Few scientists could have predicted that these tools of basic research would come to dominate the field of food diagnostics. Today, a large number of cleverly designed assay formats using these technologies are available commercially for the detection in foods of practically all major established pathogens and toxins, as well as of many emerging pathogens. These tests range from very simple antibody-bound latex agglutination assays to very sophisticated DNA amplification methods. Although molecular biological assays are more specific, sensitive, and faster than conventional (often cultural) microbiological methods, the complexities of food matrices continue to offer unique challenges that may preclude the direct application of these molecular biological methods. Consequently, a short cultural enrichment period is still required for food samples prior to analysis with these assays. The greater detection sensitivity of molecular biological methods may also affect existing microbiological specifications for foods; this undoubtedly will have repercussions on the regulatory agencies, food manufacturers, and also consumers. The US government has the right to retain a nonexclusive royalty-free license in and to any copyright covering this article. Use of trade names is for identification only and does not imply an endorsement by the US FDA.     

Prospects for chemiluminescent and bioluminescent immunoassay and nucleic acid assays in food testing and the pharmaceutical industry

The sensitivity, speed and convenience of chemiluminescent (CL) and bioluminescent (BL) immunoassays and probe assays have led to a diverse range of applications for these technologies, mainly in the clinical laboratory. These methods are now being explored by the food and pharmaceutical industries. Demanding detection limits and the complexity of sample preparation for food and pharmaceutical analyses present daunting challenges for the analyst. Immunoassay and nucleic acid amplification technologies have been applied to food testing, but these have mostly favoured non-luminescent endpoints. Food assays with CL or BL endpoints are now emerging, e.g., Clostridium botulinum type A detection using a CL immunosorbent assay; Salmonella and Zygosaccharomyces detection using a combination of PCR and CL detection. The analytical challenges posed by the pharmaceutical industry include testing for contaminants in raw materials and drug products, and drug discovery. The sensitivity and rapid signal acquisition characteristics of CL and BL are advantageous for the high throughput, massively parallel testing of micro-sized samples demanded in drug discovery. Current progress and the prospects for CL and BL immunoassay and nucleic acid technologies in this and other pharmaceutical and food applications is reviewed. © 1998 John Wiley & Sons, Ltd.     

Molecular sexing assays in 114 mammalian species: In silico sequence reanalysis and a unified graphical visualization of diagnostic tests

Molecular‐based methods for identifying sex in mammals have a wide range of applications, from embryo manipulation to ecological studies. Various sex‐specific or homologous genes can be used for this purpose, PCR amplification being a common method. Over the years, the number of reported tests and the range of tested species have increased greatly. The aim of the present analysis was to retrieve PCR‐based sexing assays for a range of mammalian species, gathering the gene sequences from either the articles or online databases, and visualize the molecular design in a uniform manner. For nucleotide alignment and diagnostic test visualization, the following genomic databases and tools were used: NCBI, Ensembl Nucleotide BLAST, ClustalW2, and NEBcutter V2.0. In the 45 gathered articles, 59 different diagnostic tests based on eight different PCR‐based methods were developed for 114 mammalian species. Most commonly used genes for the analysis were ZFX, ZFY, AMELX, and AMELY. The tests were most commonly based on sex‐specific insertions and deletions (SSIndels) and sex‐specific sequence polymorphisms (SSSP). This review provides an overview of PCR‐based sexing methods developed for mammals. This information will facilitate more efficient development of novel molecular sexing assays and reuse of previously developed tests. Development of many novel and improvement of previously developed tests is also expected with the rapid increase in the quantity and quality of available genetic information.     

Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: a review

Listeria monocytogenes is an important food-borne pathogen and is widely tested for in food, environmental and clinical samples. Identification traditionally involved culture methods based on selective enrichment and plating followed by the characterization of Listeria spp. based on colony morphology, sugar fermentation and haemolytic properties. These methods are the gold standard; but they are lengthy and may not be suitable for testing of foods with short shelf lives. As a result more rapid tests were developed based on antibodies (ELISA) or molecular techniques (PCR or DNA hybridization). While these tests possess equal sensitivity, they are rapid and allow testing to be completed within 48 h. More recently, molecular methods were developed that target RNA rather than DNA, such as RT-PCR, real time PCR or nucleic acid based sequence amplification (NASBA). These tests not only provide a measure of cell viability but they can also be used for quantitative analysis. In addition, a variety of tests are available for sub-species characterization, which are particularly useful in epidemiological investigations. Early typing methods differentiated isolates based on phenotypic markers, such as multilocus enzyme electrophoresis, phage typing and serotyping. These phenotypic typing methods are being replaced by molecular tests, which reflect genetic relationships between isolates and are more accurate. These new methods are currently mainly used in research but their considerable potential for routine testing in the future cannot be overlooked.     

Molecular typing of Vibrio parahaemolyticus isolated from seafood harvested along the south-west coast of India

Aims: Evaluation of protein profiling for typing Vibrio parahaemolyticus using 71 strains isolated from different seafood and comparison with other molecular typing techniques such as random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)‐PCR. Methods and Results: Three molecular typing methods were used for the typing of 71 V. parahaemolyticus isolates from seafood. RAPD had a discriminatory index (DI) of 0·95, while ERIC‐PCR showed a DI of 0·94. Though protein profiling had less discriminatory power, use of this method can be helpful in identifying new proteins which might have a role in establishment in the host or virulence of the organism. Conclusions: The use of protein profiling in combination with other established typing methods such as RAPD and ERIC‐PCR generates useful information in the case of V. parahaemolyticus associated with seafood. Significance and Impact of the Study: The study demonstrates the usefulness of nucleic acid and protein‐based studies in understanding the relationship between various isolates from seafood.     

DNA amplification in the field: move over PCR,here comes LAMP

It would not be an exaggeration to say that among molecular technologies, it is PCR (polymerase chain reaction) that underpins the discipline of molecular ecology as we know it today. With PCR, it has been possible to target the amplification of particular fragments of DNA, which can then be analysed in a multitude of ways. The capability of PCR to amplify DNA from a mere handful of copies further means that conservationists and ecologists are able to sample DNA unobtrusively and with minimal disturbance to the environment and the organisms of interest. However, a key disadvantage of PCR‐based methods has been the necessity for a generally non‐portable, laboratory setting to undertake the time‐consuming thermocycling protocols. LAMP (loop‐mediated isothermal amplification) offers a logistically simpler protocol: a relatively rapid DNA amplification reaction occurs at one temperature, and the products are visualized with a colour change within the reaction tubes. In the first field application of LAMP for an ecological study, Centeno‐Cuadros et al. ( 2016 ) demonstrates how LAMP can be used to determine the sex of three raptor species. By enabling DNA amplification in situ and in ‘real‐time’, LAMP promises to revolutionize how molecular ecology is practised in the field.     

Molecular community analysis of microbial diversity

New technologies that avoid the need for either gene amplification (e.g. microarrays) or nucleic acid extraction (e.g. in situ PCR) have recently been implemented in microbial ecology. Together with new approaches for culturing microorganisms and an increased understanding of the biases of molecular methods, these techniques form the most exciting advances in this field during the past year.     

A comparison of nucleic acid amplification techniques for the assessment of bacterial viability

AIMS: The ability to determine the presence and viability status of bacteria by molecular methods could offer significant advantages to the food, environmental and health sectors, in terms of improved speed and sensitivity of detection. METHODS AND RESULTS: In this study, we have assessed three amplification techniques, PCR, RT-PCR and NASBA, for their ability to detect nucleic acid persistence in an E. coli strain following heat-killing. NASBA offered the greatest sensitivity of the three methods tested. The presence of residual DNA and mRNA could be detected by PCR and NASBA, respectively, for up to 30 h postdeath, by which time cell death had been confirmed by culture methods. Thus a single quantitative measurement based on nucleic acid amplification did not permit unequivocal determination of cell viability. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation between cell viability and persistence of nucleic acids must be well characterized for a particular analytical situation before molecular techniques can be substituted for traditional culture methods.     

Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field

The increase in foodborne outbreaks highlights the need for rapid, sensitive and specific methods for food safety monitoring, enabling specific detection and quantification of viable foodborne pathogens. Real‐time PCR (qPCR) combined with the use of viability dyes, recently introduced, fulfils all these requirements. The strategy relies on the use of DNA‐binding molecules such as propidium monoazide (PMA) or ethidium monoazide (EMA) as sample pretreatment previous to the qPCR. These molecules permeate only membrane‐compromised cells and have successfully been applied for different types of foodborne pathogens, including bacteria and viruses. Moreover, those dyes have been explored to monitor different food manufacturing processes as an alternative to classical cultural methods. In this review, state‐of‐the‐art information regarding viability PCR (v‐PCR) is compiled.     

数字PCR技术及其在检测领域的应用

随着分子生物学技术的不断发展和需求的多样化,用于核酸检测的各种PCR衍生技术应运而生。数字PCR是一种单分子水平的大规模分区扩增定量核酸检测技术。该技术以微腔室/微孔或微滴作为PCR反应器,无需校准物和绘制标准曲线即可实现对样品初始浓度的绝对定量,具有高灵敏度、高特异性和高精确度的特点。本文详细介绍了数字PCR的技术发展史、作用原理以及仪器平台类型,系统阐述了数字PCR在转基因检测、疾病诊断、环境及食品监管等方面的应用概况,并对该技术的应用前景进行了展望,以期对未来数字PCR的开发利用提供参考。