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.     

Computational approaches in design of nucleic acid-based therapeutics

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核酸诊断技术规范化的研究

针对PCR技术应用中的一些问题,从下述几方面开展了核酸诊断技术标准化的研究:①探讨了设计引物的影响因素,发现根据同一基因设计的不同引物对PCR敏感性影响较大,可相差20倍,进一步研究表明,引物的自由能,尤其是引物3'端6个碱基的自由能可能决定着PCR的敏感性;②研制成功室温稳定的PCR试剂,将所有PCR成分(加入一种酶保护剂)配制并分装于微量离心管中,并干燥,试剂室温放置8个月,37℃破坏2周对检测结果无任何影响;③因尿嘧啶糖基化酶(UDG)可特异降解DNA双链中的尿密啶核苷,PCR体系中以dUTP代替dTTP,并在PCR扩增前用UDG消化15 min,就可以特异防止PCR产物的污染.对炭疽杆菌、鼠疫杆菌、土拉杆菌和布鲁氏菌扩增结果表明,0.1 U的UDG可完全消化103～108个产物分子的污染;④建立了微孔板杂交技术代替电泳方法检测扩增产物,将PCR产物克隆作为捕获探针包被聚乙烯微孔板,PCR引物5'末端生物素标记,扩增后作为待检测探针杂交,通过比较影响微孔板杂交的包被、杂交和显色等因素,建立了PCR-EIA技术.通过比较包被缓冲液发现镁离子及其离子强度是影响DNA包被的重要因素,包被的DNA以线型质粒最佳,每孔包被300ng左右的DNA 杂交效果最好;杂交液中加和不加甲酰胺对杂交影响不大;用链霉亲和素化的辣根过氧化物酶或碱性磷酸酶酶联显色均能达到检测杂交体的目的,但前者显色较快,更为实用.确定了PCR-EIA技术的最佳的包被、杂交和显色的条件,比常规的PCR-电泳检测敏感性高10倍;⑤比较了不同标本处理方法,确定了各种临床标本和动物组织的较为理想的处理方法.通过上述不同方面的研究,确定了从引物设计、试剂配制、标本处理、UDG防污染和产物微孔板杂交-酶联显色检测等方面的优化条件,建立起敏感性高、特异性强、操作简便和检测客观的核酸检测技术,为临床实验室的常规应用和反生物战病原微生物的核酸诊断奠定了基础.     

Synthetic circuits,devices and modules

The aim of synthetic biology is to design artificial biological systems for novel applications. From an engineering perspective, construction of biological systems of defined functionality in a hierarchical way is fundamental to this emerging field. Here, we highlight some current advances on design of several basic building blocks in synthetic biology including the artificial gene control elements, synthetic circuits and their assemblies into devices and modules. Such engineered basic building blocks largely expand the synthetic toolbox and contribute to our understanding of the underlying design principles of living cells.     

Application of molecular nucleic acid-based techniques for the study of microbial communities in monuments and artworks.

Microorganisms play critical roles in every kind of habitat on Earth, including those constructed by humans. Thus, our cultural heritage is affected by microbial colonization. While classical microbiological methods based on culturing procedures have provided important, but limited information on the microbial diversity of natural samples, novel molecular techniques have been extremely valuable in unraveling the diversity of microbiota involved in the biodeterioration of our monuments and artworks. The knowledge gained from these approaches has allowed the design of strategies for conserving and protecting monuments for the benefit of future generations. This review describes the state-of-the-art of the application of molecular methods to the analysis of cultural assets, and provides near-future perspectives on the subject.     

Plant pathogen diagnostics: immunological and nucleic acid-based approaches

Accurate disease diagnosis and precise identification of any pathogens involved is an essential prerequisite for understanding plant diseases and controlling them effectively. Traditional methods of identifying plant pathogens can be slow and inconclusive, and this has prompted the search for alternative diagnostic techniques. Here, we review recent developments in antibody-based and nucleic acid-based diagnostic methods, and their applications in plant pathology. We also highlight some new developments in diagnostics that are likely to make an impact in the future.     

A rational approach in probe design for nucleic acid-based biosensing

Development of nucleic acid-based sensing attracts the interest of many researchers in the field of both basic and applied research in chemistry. Major factors for the fabrication of a successful nucleic acid sensor include the design of probes for target sequence hybridization and their immobilization on the chip surface. Here we demonstrate that a rational choice of bioprobes has important impact on the sensor's analytical performances. Computational evaluations, by a simple and freely available program, successfully led to the design of the best probes for a given target, with direct application to nucleic acid-based sensing. We developed here an optimized and reproducible strategy for in silico probe design supported by optical transduction experiments. In particular Surface Plasmon Resonance imaging (SPRi), at the forefront of optical sensing, was used here as proof of principle. Five probes were selected, immobilized on gold chip surfaces by widely consolidated thiol chemistry and tested to validate the computational model. Using SPRi as the transducting component, real-time and label free analysis was performed, taking the Homo sapiens actin beta (ACTB) gene fragment as model system in nucleic acid detection. The experimental sensor behavior was further studied by evaluating the strength of the secondary structure of probes using melting experiments. Dedicated software was also used to evaluate probes' folding, to support our criteria. The SPRi experimental results fully validate the computational evaluations, revealing this approach highly promising as a useful tool to design biosensor probes with optimized performances.     

A nucleic acid-based test for detection of Fasciola hepatica.

The use of nucleic acid techniques in the diagnosis of parasitic infection has become increasingly widespread. An oligonucleotide probe derived from a rRNA sequence was developed for the detection of Fasciola hepatica in its intermediate snail host Pseudosuccinea columella. Total RNA obtained from whole adult liver flukes was used in a polymerase chain reaction to isolate and amplify a region of approximately 650 base pairs in the small subunit rRNA. This portion of the ribosomal cDNA, which contains highly conserved regions as well as variable regions, was subcloned and sequenced. In comparison to known small subunit rRNA sequences, a sequence unique to F. hepatica was identified and an oligonucleotide probe (CS4) for detection of F. hepatica was developed. A northern blot analysis using CS4 successfully identified small subunit rRNA from F. hepatica. Slot-blot analysis determined that RNA derived from 5 miracidia can be detected with CS4. Moreover, a slot blot utilizing CS4 distinguished RNA derived from snails infected with F. hepatica from RNA of uninfected snails.     

Synthesis and antiviral evaluation of nucleic acid-based (NAB) libraries

Combinatorial assembly of nucleotide libraries and their antiviral evaluation against HSV-1 are described.     

Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections

Here we describe a method for sensitive and specific histological detection of microRNAs (miRNAs) by in situ hybridization. The protocol focuses on the use of locked nucleic acids (LNAs), which are bi-cyclic RNA analogs that allow a significant increase in the hybridization temperature and thereby an enhanced stringency for short probes as required for miRNA detection. The protocol is optimized for cryosections in order to study the spatial and temporal expression of miRNAs with high sensitivity and resolution. We detail how to construct probes, set up and conduct an LNA in situ hybridization experiment. In addition, we discuss alternative colorimetric strategies that can be used to effectively detect and visualize miRNAs including double staining with other markers. Setting up and conducting the in situ experiment is estimated to take approximately 1 week, assuming that all the component parts are readily available.     

Rapid and sensitive detection of Taura syndrome virus using nucleic acid-based amplification

Requiring only simple heating devices, isothermal nucleic acid-based amplification (NASBA) is a potential detection platform to be developed for on-site diagnosis of aquaculture pathogens. In this report, an NASBA assay has been developed for the Taura syndrome virus (TSV), one of the most devastating RNA virus pathogens for several penaeid shrimp species. The NASBA amplicons were detected by agarose gel electrophoresis and confirmed by Northern-blotting and dot-blotting analysis, using a biotinylated TSV-specific primer. The sensitivity of the TSV NASBA coupled with dot-blotting detection was approximately 5-fold less sensitive than that of the commercially available RT-nested, PCR-based IQ2000 TSV Detection and Prevention System that was also confirmed to be more sensitive than the RT-PCR-based TSV detection protocol recommended by the OIE (Office International des Epizooties). The specificity of the TSV NASBA reaction was substantiated by the results that RNA of non-target viruses did not generate any signals. Furthermore, a simple colorimetric microtiter plate assay employing TSV-specific capture and detection primers was developed as a simple alternative approach for the detection of NASBA amplicons. Taken together, the combination of the isothermal NASBA and colorimetric solid phase-based assays should allow sensitive, straightforward, and speedy on-site detection of TSV.     

Dual properties of the deacetylated sites in chitosan for molecular immobilization and biofunctional effects

Polypropylene nonwoven fabric was surface-activated by high-density oxygen microwave plasma, followed by graft copolymerization with acrylic acid (AAc) and then coupling with chitosan molecules. The pAAc-grafted surface containing C=O in carboxylic acid exhibited a hydrophilic character capable of promoting water absorbency. A larger portion of minimum 85% deacetylated sites in chitosan molecules was then coupled with the grafted pAAc (around 149 microg.cm(-2)) by forming amide bonds at their interface. The covalently bonded chitosan was weighted around 44 microg.cm(-2). The smaller portion of the deacetylated sites demonstrated a distinctive structure as polycations, i.e., NH(3)(+), on the immobilized chitosan. The respective structures following sequential reactions were identified using Fourier transform infrared-attenuated total reflection and X-ray photoelectron spectroscopy with peaks deconvolution. The NH(3)(+) sites on the immobilized chitosan exhibited biofunctional in anticoagulation and in antibacterial property. Blood cells agglutination or agglomeration upon the chitosan-immobilized surface, in particular for red blood cells and platelets, resulted from hydrophilic effect derived from the grafted pAAc and the chitosan itself, and ionic attractions between polycations and blood cells. In addition, the agglutinated cells retained their original morphologies. It is therefore very promising to apply this durable chitosan-immobilized surface for making an antibacterial support, at the same time, for retaining blood cell affinity.     

Comparison of nucleic acid-based detection of avian influenza H5N1 with virus isolation

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) of avian influenza virus was compared with viral culture in embryonated chicken eggs. Virus was isolated from blood or anal swabs of chickens artificially infected with highly pathogenic avian influenza A/Chicken/Hong Kong/1000/97 (H5N1). Viral nucleic acid was detected in blood samples by NASBA/ECL immediately prior to death, whilst nucleic acid extracted from anal swabs was detected from the day following artificial infection until death. Thus, blood and/or anal swabs are a suitable source of material for the detection of avian influenza in dead birds, but anal swabs are more suitable for detection of viral genetic material in live birds. Dilution of a known viral standard was used to determine the limit of sensitivity for both NASBA/ECL and egg culture detection methods. The NASBA/ECL method was equivalent in sensitivity to egg culture. The NASBA/ECL results agreed with egg culture data in 71/94 (75.5%) tissue samples obtained from artificially infected birds.     

Generation of monoclonal antibodies against prion proteins with an unconventional nucleic acid-based immunization strategy.

Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI). The pathomechanisms of the prion diseases are not yet understood. Therefore, monoclonal antibodies (mAbs) would provide valuable tools in diagnostics as well as in basic research of these diseases. In contrast to conventional strategies we have developed an immunization protocol based on nucleic acid injection into non tolerant PrP0/0-mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS and FFI were injected into muscle tissue. The mice were primarily inoculated with DNA-plasmids encoding PRNP and boosted either with DNA, RNA or recombinant Semliki Forest virus (SFV) particles expressing PRNP. After hybridoma preparation, different mAbs against prion proteins were obtained and their binding behaviour was analysed by peptide-ELISA, Western blot, immunofluorescence and immunoprecipitation. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. It could, therefore, be demonstrated that immunization of non tolerant mice with DNA and live attenuated SF virus is a valuable means to induce a broad immune response leading eventually to the generation of a panel of mAbs for basic science as well as for diagnostics.