Innovative tools for detection of plant pathogenic viruses and bacteria

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.     

PNA for rapid microbiology.

The acceptance of rRNA sequence diversity as a criterion for phylogenetic discrimination heralds the transition from microbiological identification methods based on phenotypic markers to assays employing molecular techniques. Robust amplification assays and sensitive direct detection methods are rapidly becoming the standard protocols of microbiology laboratories. The emergence of peptide nucleic acid (PNA) from its status as an academic curiosity to that of a promising and powerful molecular tool, coincides with, and complements, the transition to rapid molecular tests. The unique properties of PNA enable the development of assay formats, which go above and beyond the possibilities of DNA probes. PNA probes targeting specific rRNA sequences of yeast and bacteria with clinical, environmental, and industrial value have recently been developed and applied to a variety of rapid assay formats. Some simply incorporate the sensitivity and specificity of PNA probes into traditional methods, such as membrane filtration and microscopic analysis; others involve recent techniques such as real-time and end-point analysis of amplification reactions.     

A sensitive branched DNA HIV-1 signal amplification viral load assay with single day turnaround

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) (“Versant Assay”) currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16–18 h to 2.5 h, composition of only the “Lysis Diluent” solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements.     

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.     

滚环扩增技术的原理及其应用的研究

滚环扩增是近年来发展起来的一种恒温核酸扩增方法。这种方法不仅可以直接扩增DNA和RNA,还可以实现对靶核酸的信号放大,灵敏度达到一个拷贝的核酸分子,因此,RCA技术在全基因组扩增、单核苷酸多态性、DNA芯片、蛋白质芯片等方面检测中具有很大的应用价值和潜力。     

Enzyme immunoassays for the detection of microbial antigens and prospects for improved assays

The rapid diagnosis of viral infections is an important tool in the management of patients with infectious diseases. Solid-phase enzyme immunoassays have proved to be useful tools for the direct detection of the antigens of some viruses directly in clinical specimens. Such assays have been particularly useful in the diagnosis of viral infections in the gastrointestinal and respiratory tracts. However, standard solid-phase enzyme immunoassays often do not display sufficient sensitivity for the diagnosis of all cases of viral infections. Techniques which might be utilized to increase the sensitivity of solid-phase immunoassays include the use of monoclonal antibodies to maximize the efficiency of the antigen-antibody interactions and the use of high-turnover enzymes to increase the amount of signal generated by the ensuing enzyme-substrate reactions. In addition, techniques making use of nucleic acid hybridization have a great deal of potential for the accurate detection of viral nucleic acids in human body fluids. The successful application of these techniques to the diagnosis of viral infections could lead to a marked improvement in the care of patients with suspected infectious diseases as well as to a decrease in the transmission of viral infections to high-risk individuals.     

Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application

Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.     

Locked nucleic acid flow cytometry-fluorescence in situ hybridization (LNA flow-FISH): a method for bacterial small RNA detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination, cellular identification and gene expression, monitoring viral multiplication in infected cells, and bacterial community analysis and enumeration. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (T(m)) of these analogs for natural nucleic acids. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes and have been successfully used to detect eukaryotic mRNA and viral RNA in mammalian cells. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).     

基因扩增产物的固相杂交-酶联显色方法的建立

建立基于基因扩增技术的简便、快速的病毒核酸定量检测方法．将标记有生物素的寡核苷酸引物所扩增的病毒基因产物,与通过共价键结合在微孔反应板上的特异性探针进行快速杂交,然后通过辣根过氧化物酶标记的抗生物素进行酶联显色,读取光密度值．应用本方法对血清中乙型、丙型肝炎病毒核酸定量检测,灵敏度分别可达１－５拷贝／反应．此方法简便、快速、特异性好、敏感性高、半定量指标客观,可广泛应用于肝炎病毒感染的临床诊断和疗效评价．     

Hybridocytochemistry with non-radioactive probes as a tool for biomedical research and cytodiagnostics

The advent of DNA and RNA labelling techniques which allow non-autoradiographic detection of specific nucleic acid sequences has opened new possibilities for rapid diagnosis at the nucleic acid level. There are already practical applications. However, in order to fully compete with radioactive procedures an increase in sensitivity is still desired.     

Target-Dependent Enrichment of Virions Determines the Reduction of High-Throughput Sequencing in Virus Discovery

Viral infections cause many different diseases stemming both from well-characterized viral pathogens but also from emerging viruses, and the search for novel viruses continues to be of great importance. High-throughput sequencing is an important technology for this purpose. However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue. Techniques to enrich viral targets in high-throughput sequencing have been reported, but the sensitivity of such methods is not well established. This study compares different library preparation techniques targeting both DNA and RNA with and without virion enrichment. By optimizing the selection of intact virus particles, both by physical and enzymatic approaches, we assessed the effectiveness of the specific enrichment of viral sequences as compared to non-enriched sample preparations by selectively looking for and counting read sequences obtained from shotgun sequencing. Using shotgun sequencing of total DNA or RNA, viral targets were detected at concentrations corresponding to the predicted level, providing a foundation for estimating the effectiveness of virion enrichment. Virion enrichment typically produced a 1000-fold increase in the proportion of DNA virus sequences. For RNA virions the gain was less pronounced with a maximum 13-fold increase. This enrichment varied between the different sample concentrations, with no clear trend. Despite that less sequencing was required to identify target sequences, it was not evident from our data that a lower detection level was achieved by virion enrichment compared to shotgun sequencing.     

Immobilon-Ny+ nucleic acid blotting membrane: an advanced nylon membrane optimized for superior fixation and reprobing

The use of charged nylon membranes in nucleic acid blotting applications has become an important factor in the success of hybridization-based assays. Retention of nucleic acids on these membranes is promoted by baking at 80 degrees C under vacuum or by exposure to short wavelength UV light, with the latter method preferred. Immobilon-Ny+ is an advanced, positively charged nylon membrane that has been optimized to show superior retention of target DNA and RNA under hybridization conditions. Higher signal levels are obtained in these assays compared to competitive membranes, even after 13 cycles of probing. This report illustrates the superior performance of Immobilon-Ny+ in 32P and chemiluminescent hybridization assays on blotted DNA and RNA.     

Ligation-based molecular tools for lab-on-a-chip devices

Molecular diagnostics can offer early detection of disease, improved diagnostic accuracy, and qualified follow-up. Moreover, the use of microfluidic devices can in principle render these analyses quickly and user-friendly, placing them within the reach of the general practitioner and maybe even in households. However, the progress launching such devices has been limited so far. We propose that an important limiting factor has been the difficulty of establishing molecular assays suitable for microfabricated formats. The assays should be capable of monitoring a wide range of molecules, including genomic DNA, RNA and proteins with secondary modifications and interaction partners, and they must exhibit excellent sensitivity and specificity. We discuss these problems and describe a series of molecular tools that may present new opportunities for lab-on-a-chip devices at the point-of-care.     

Quantification of DNA sequences obtained by polymerase chain reaction using a bioluminescent adsorbent.

We studied various parameters affecting the sensitivity of assays that use nucleic acid hybridization in solution followed by capture of the hybrid on a solid phase. Sensitivity is limited not only by nonspecific binding of the detection components but also by reannealing of the target or probe to itself. To perform sensitive assays, the probe concentration must be low enough to reduce high nonspecific binding. Under these conditions, however, the strand displacement reaction or the reannealing of the target to itself drastically decreases the hybridization yield, particularly when the target and the probes are different sizes. To improve DNA detection, we propose a sandwich method based on hybridization of oligonucleotides with a single-strand DNA obtained by polymerase chain reaction under asymmetric conditions. The assay can be performed in one step using a bioluminescent detection procedure which does not require any separation step. The specificity of the method is sufficient to perform a rapid detection and quantification of papillomavirus in biological samples.     

Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations

Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. The ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).     

Quantitation of viral load using real-time amplification techniques

Real-time PCR amplification techniques are currently used to determine the viral load in clinical samples for an increasing number of targets. Real-time PCR reduces the time necessary to generate results after amplification. In-house developed PCR and nucleic acid sequence-based amplification (NASBA)-based systems combined with several detection strategies are being employed in a clinical diagnostic setting. The importance of these assays in disease management is still in an exploration phase. Although these technologies have the implicit capability of accurately measuring DNA and RNA in clinical samples, issues related to standardization and quality control must be resolved to enable routine implementation of these technologies in molecular diagnostics.