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。     

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、装备要求低以及可现场实时检测等方面具有明显的技术优势,已成为食源性病毒检测领域的热点研究方向。因此,本文对近年来食源性病毒核酸恒温检测技术的原理、应用、优缺点等方面进行综述,并对未来发展方向进行了展望。     

DNA生物传感器在环境污染监测中的应用

基于生物催化和免疫原理的生物传感器在环境领域中获得了广泛应用.近年来,随着分子生物学和生物技术的发展,人们开发了以核酸探针为识别元件,基于核酸相互作用原理的DNA生物传感器.该传感器可用于受感染微生物的核酸序列分析、优先控制污染物的检测以及污染物与DNA之间相互作用的研究,在环境污染监测中具有潜在的巨大应用前景.简要介绍了核酸杂交生物传感器的基本原理及其在环境微生物和优先控制污染物（priority pollutant）检测中的应用研究进展.     

FEN1酶介导的功能核酸生物传感器的研究进展

瓣状核酸内切酶-1(Flap endonuclease 1,FEN1)是一种可以识别三碱基重叠结构(三核酸)并对其进行切割,释放出5’-flap片段的结构特异性酶,并且有着高效稳定的切割效率。基于此种特性,通过不同的信号输出方式,FEN1酶现被用于DNA、RNA、病毒等放大检测中。首先对FEN1酶的发现、性质以及作用方面做了相应介绍,然后根据所检测的靶物质不同,对FEN1酶所介导的生物传感器进行分类,主要包括对单核苷酸多态性的检测、甲基化检测、基因型检测、RNA检测、病毒检测、肿瘤检测和微生物检测等。此外,对FEN1酶与纳米材料的结合以及体内表征及治疗也进行了较为详细的介绍。同时,还对传感器之间的原理、灵敏度、特异性及适用领域等方面进行比较和优缺点的简单评价。最后,对FEN1酶所介导的生物传感器的中存在的不足,以及未来的发展方向进行了展望,旨在为今后研发更便携、更灵敏、更准确的FEN1功能核酸生物传感器提供理论参考。     

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.     

功能核酸DNA水凝胶的制备与组装

功能核酸DNA水凝胶是一种以DNA为构建单元通过化学反应或物理缠结自组装而成的新型柔性材料,其构建单元中包含1种或多种能够形成功能核酸的特定序列。功能核酸是通过碱基修饰和DNA分子之间的相互作用力组合的一类特定核酸结构,包括核酸适配体、DNA核酶、G-四联体(G-quadruplex,G4)和i-motif结构等。传统上,高浓度的长DNA链是制备DNA水凝胶的必要条件,而核酸扩增方法的引入为DNA水凝胶的组装方式提供了新的可能。因此,对常用于制备DNA水凝胶的多种功能核酸以及核酸的提取、合成和扩增手段进行了详细的介绍。在此基础上,综述了通过化学或物理交联方式组装功能核酸DNA水凝胶的制备方法。最后,提出了DNA纳米材料的组装所面临的挑战和潜在的发展方向,以期为开发高效组装的功能核酸DNA水凝胶提供参考。     

A universal platform for sensitive and selective colorimetric DNA detection based on Exo III assisted signal amplification

Rapid growth of available sequence data has made the detection of nucleic acids critical to the development of modern life sciences. Many amplification methods based on gold nanoparticles and endonuclease for sensitive DNA detection have been developed. However, these approaches require specific target sequence for endonuclease recognition, which cannot be fulfilled in all systems. Replacing the restriction enzyme with a nuclease that does not require any specific recognition sequence may offer a universally adaptable system. Here we have developed a novel homogeneous, colorimetric DNA detection method, which consists of Exo III, a linker DNA, and two DNA-modified gold nanoparticles. This system is simple, low-cost, sensitive and selective. By coupling cyclic enzymatic cleavage and gold nanoparticle for signal amplification, our system provides a colorimetric detection limit of 15 pM, which is 3 orders of magnitude more sensitive than that of a general three-component sandwich assay format. Due to the intrinsic property of Exo III, our method shows excellent detection selectivity for single-base discrimination. More importantly, superior to other methods based on nicking and FokI endonuclease, our target sequence-independent platform is generally applicable for DNA sensing. This new approach could be widely applied to sensitive nucleic acids detection.     

Ultrasensitive nucleic acid biosensors for early cancer diagnostics

We have developed ultrasensitive nucleic acid detection systems involving an amplification step where the analytical signal correlates directly to the amount of nucleic acid in the solution So far, we have performed nucleic acid quantification on several breast cancer susceptibility genes and were able to detect nucleic acid amounts that ranged from 0.1–1.0 fg of nucleic acid, which is at least 1000 times more sensitive than conventional fluorescent detection methods. The biosensors are so sensitive that they can be used for direct detection of breast cancer susceptibility genes in mRNA without involving a PCR step.     

Detection of viral infection and gene expression in clinical tissue specimens using branched DNA (bDNA) in situ hybridization.

In situ hybridization (ISH) methods for detection of nucleic acid sequences have proved especially powerful for revealing genetic markers and gene expression in a morphological context. Although target and signal amplification technologies have enabled researchers to detect relatively low-abundance molecules in cell extracts, the sensitive detection of nucleic acid sequences in tissue specimens has proved more challenging. We recently reported the development of a branched DNA (bDNA) ISH method for detection of DNA and mRNA in whole cells. Based on bDNA signal amplification technology, bDNA ISH is highly sensitive and can detect one or two copies of DNA per cell. In this study we evaluated bDNA ISH for detection of nucleic acid sequences in tissue specimens. Using normal and human papillomavirus (HPV)-infected cervical biopsy specimens, we explored the cell type-specific distribution of HPV DNA and mRNA by bDNA ISH. We found that bDNA ISH allowed rapid, sensitive detection of nucleic acids with high specificity while preserving tissue morphology. As an adjunct to conventional histopathology, bDNA ISH may improve diagnostic accuracy and prognosis for viral and neoplastic diseases.     

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.     

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.     

Isothermal amplification detection of nucleic acids by a double-nicked beacon

Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use.     

基于CRISPR/Cas系统的纸基分析在快速检测食源性致病菌中的应用

由食源性致病菌引起的食品安全事件严重影响人类健康,开发针对食源性致病菌的快速检测技术十分必要。成簇间隔短回文重复序列（clustered regularly interspaced short palindromic repeats,CRISPR）及相关蛋白（CRISPR-associated protein,Cas）是原核生物的适应性免疫系统,具有特异性识别并切割核酸序列的功能。纸基分析方法作为一种简便性好、成本低廉的分析检测工具,在快速检测领域展现出良好的前景。因此,将CRISPR/Cas系统的高效识别能力和纸基分析方法的简便性相结合可实现对食源性致病菌的快速灵敏检测。本文简要介绍了CRISPR/Cas系统用于核酸检测的概况,对第二类单Cas效应蛋白系统的特点及原理进行概述,重点综述基于CRISPR/Cas系统的试纸分析、侧向流动分析和纸基微流控装置在检测食源性致病菌方面的应用,并讨论了CRISPR/Cas系统结合纸基分析建立检测方法的优势、当前的挑战及未来的发展前景。     

A decade with nucleic acid‐based microbiological methods in safety control of foods

In the last decade, nucleic acid‐based methods gradually started to replace or complement the culture‐based methods and immunochemical assays in routine laboratories involved in food control. In particular, real‐time polymerase chain reaction (PCR) was technically developed to the stage of good speed, sensitivity and reproducibility, at minimized risk of carry‐over contamination. Basic advantages provided by nucleic acid‐based methods are higher speed and added information, such as subspecies identification, information on the presence of genes important for virulence or antibiotic resistance. Nucleic acid‐based methods are attractive also to detect important foodborne pathogens for which no classical counterparts are available, namely foodborne pathogenic viruses. This review briefly summarizes currently available or developing molecular technologies that may be candidates for involvement in microbiological molecular methods in the next decade. Potential of nonamplification as well as amplification methods is discussed, including fluorescent in situ hybridization, alternative PCR chemistries, alternative amplification technologies, digital PCR and nanotechnologies.     

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.     

基于CRISPR/Cas体系的生物传感器在病原体核酸检测方面的应用

成簇规律间隔短回文重复序列/成簇规律间隔短回文重复序列相关蛋白 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein,CRISPR/Cas) 因高效的靶向结合和可编程性,已被开发为一种精准、高效、低价和高灵敏度的核酸检测工具。目前基于CRISPR-Cas体系的生物传感器在病原体核酸检测方面显示出了优良的性能,受到了人们的广泛关注,这种新型的病原体核酸检测有望替代传统的病原体检测方法。文中就基于CRISPR/Cas体系的生物传感器在病原体核酸检测中的最新研究进展进行综述。     

Postcolumn nucleic acid intercalation for the fluorescent detection of nucleic acids using ion pair reverse phase high-performance liquid chromatography

Here we report a simple and effective procedure enabling the fluorescent detection of nucleic acids following the rapid, high-resolution separation using ion pair reverse phase chromatography. This approach uses postcolumn nucleic acid intercalation of fluorescent dyes with subsequent fluorescent detection, demonstrating more than a 1000-fold increase in sensitivity in the detection of nucleic acids when compared with traditional UV detection. Moreover, a wide range of intercalating dyes can be incorporated, including those known to disrupt the structure of the nucleic acids, thereby enabling the sensitive detection of DNA and RNA with no adverse effect on resolution of the nucleic acids during ion pair reverse phase chromatography. In addition, such approaches allow one to readily distinguish single-stranded DNA from double-stranded DNA following their separation using ion pair reverse phase high-performance liquid chromatography.     

纳米粒子标记DNA 探针在电化学DNA 生物传感器中的应用

介绍了纳米电化学DNA生物传感器的基本概念和分类,并介绍了用于DNA标记的纳米粒子的六种类型及其三大检测方法,在此基础上对纳米电化学DNA生物传感器在基因检测、疾病诊断、DNA检测等方面的最新进展进行了综述与讨论。