Simultaneous Detection Of DNA Research Articles

Development of a multiplex RT-RPA assay for simultaneous detection of three viruses in cucurbits.

Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) on cucurbit crops has been reported to cause devastating crop losses in many regions of the world. In this study, a multiplex recombinase polymerase amplification (RPA) assay, an isothermal technique for rapid and simultaneous detection of DNA and RNA viruses CuLCrV, CYSDV and CCYV was developed. Highly specific and sensitive multiplex RPA primers for the coat protein region of these viruses were created and evaluated. The sensitivity of the multiplex RPA assay was examined using serially diluted plasmid containing the target regions. The results demonstrated that multiplex RPA primers have high sensitivity with a detection limit of a single copy of the viruses. The multiplex RPA primers were specific to the target as indicated by testing against other begomoviruses, potyviruses and an ilarvirus, and no nonspecific amplifications were noted. The primers simultaneously detected mixed infection of CCYV, CYSDV and CuLCrV in watermelon and squash crude extracts. This study is the first report of a multiplex RPA assay for simultaneous detection of mixed infection of DNA and RNA plant viruses.

Comparison and development of a metagenomic next-generation sequencing protocol for combined detection of DNA and RNA pathogens in cerebrospinal fluid

BackgroundThe purpose of this study was to evaluate different pretreatment, extraction, amplification, and library generation methods for metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) and to develop an efficient procedure for the simultaneous detection of DNA and RNA pathogens.MethodsWe generated thirteen mock CSF samples with four representative pathogens of encephalitis. Each sample was subjected to ten different methods by varying sample pretreatment/nucleic acid extraction (microbial DNA, total DNA, total NA, total RNA, Whole Transcriptome Amplification (WTA)) and library generation (Illumina or NEB). Negative extraction controls (NECs) were used for each method variation.ResultsWe found that the quality of mNGS sequencing reads was higher from the NEB kit for library generation. Microbial DNA and total RNA increased microbial deposition by depleting the host DNA. Methods total NA and total RNA can detect gram-positive, gram-negative, RNA and DNA pathogens. We applied mNGS, including total NA and NEB library generation, to CSF samples from five patients diagnosed with infectious encephalitis and correctly determined all pathogens identified in clinical etiological tests.ConclusionsOur findings suggested that total nucleic acid extraction combined with NEB library generation is the most effective mNGS procedure in CSF pathogen detection. The optimization of positive criteria and databases can improve the specificity and sensitivity of mNGS diagnosis.Trial registration: Chinese Clinical Trial Registry, ChiCTR1800015425 (29/03/2018), https://www.chictr.org.cn/edit.aspx?pid=26292&htm=4.

Simultaneous detection and quantification of DNA and protein biomarkers in spectrum of cardiovascular diseases in a microfluidic microbead chip

The rapid and simultaneous detection of DNA and protein biomarkers is necessary to detect the outbreak of a disease or to monitor a disease. For example, cardiovascular diseases are a major cause of adult mortality worldwide. We have developed a rapidly adaptable platform to assess biomarkers using a microfluidic technology. Our model mimics autoantibodies against three proteins, C-reactive protein (CRP), brain natriuretic peptide (BNP), and low-density lipoprotein (LDL). Cell-free mitochondrial DNA (cfmDNA) and DNA controls are detected via fluorescence probes. The biomarkers are covalently bound on the surface of size- (11–15 μm) and dual-color encoded microbeads and immobilized as planar layer in a microfluidic chip flow cell. Binding events of target molecules were analyzed by fluorescence measurements with a fully automatized fluorescence microscope (end-point and real-time) developed in house. The model system was optimized for buffers and immobilization strategies of the microbeads to enable the simultaneous detection of protein and DNA biomarkers. All prime target molecules (anti-CRP, anti-BNP, anti-LDL, cfmDNA) and the controls were successfully detected both in independent reactions and simultaneously. In addition, the biomarkers could also be detected in spiked human serum in a similar way as in the optimized buffer system. The detection limit specified by the manufacturer is reduced by at least a factor of five for each biomarker as a result of the antibody detection and kinetic experiments indicate that nearly 50 % of the fluorescence intensity is achieved within 7 min. For rapid data inspection, we have developed the open source software digilogger, which can be applied for data evaluation and visualization.Graphical abstract

Dual-colored graphene quantum dots-labeled nanoprobes/graphene oxide: functional carbon materials for respective and simultaneous detection of DNA and thrombin

Convenient and simultaneous detection of multiple biomarkers such as DNA and proteins with biocompatible materials and good analytical performance still remains a challenge. Herein, we report the respective and simultaneous detection of DNA and bovine α-thrombin (thrombin) entirely based on biocompatible carbon materials through a specially designed fluorescence on-off-on process. Colorful fluorescence, high emission efficiency, good photostability and excellent compatibility enables graphene quantum dots (GQDs) as the best choice for fluorophores in bioprobes, and thus two-colored GQDs as labeling fluorophores were chemically bonded with specific oligonucleotide sequence and aptamer to prepare two probes targeting the DNA and thrombin, respectively. Each probe can be assembled on the graphene oxide (GO) platform spontaneously by π–π stacking and electrostatic attraction; as a result, fast electron transfer in the assembly efficiently quenches the fluorescence of probe. The presence of DNA or thrombin can trigger the self-recognition between capturing a nucleotide sequence and its target DNA or between thrombin and its aptamer due to their specific hybridization and duplex DNA structures or the formation of apatamer–substrate complex, which is taken advantage of in order to achieve a separate quantitative analysis of DNA and thrombin. A dual-functional biosensor for simultaneous detection of DNA and thrombin was also constructed by self-assembly of two probes with distinct colors and GO platform, and was further evaluated with the presence of various concentrations of DNA and thrombin. Both biosensors serving as a general detection model for multiple species exhibit outstanding analytical performance, and are expected to be applied in vivo because of the excellent biocompatibility of their used materials.

Simultaneous detection of DNA from 10 food allergens by ligation-dependent probe amplification

The simultaneous detection of DNA from different allergenic food ingredients by a ligation-dependent probe amplification (LPA) system is described. The approach allows detection of several targets in a one-tube assay. Synthetic oligonucleotides were designed to detect DNA from peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts. The specificity of the system was tested with DNA from more than 50 plant and animal species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg kg−1 range. The limit of detection (LOD) for single allergens in different food matrices was 5 mg kg−1. The novel analytical strategy represents a useful tool for the surveillance of established legislation on food allergens within the European Union.

Multifunctional Electrode Arrays: Towards a Universal Detection Platform

AbstractIn this article we review recent work in our laboratory towards the realization of a multianalyte microelectrode detection platform capable of discriminating chemicals and different biomolecules simultaneously. The functionalization of electrodes with aryl diazonium salts provides an electrically addressable deposition procedure capable of immobilizing a wide range of molecules. We demonstrate control over surface density and electron transfer kinetics as well as the activation of individual electrodes in an array. The direct electrically‐addressable immobilization of diazonium‐modified proteins is shown to be suitable for the construction of multianalyte immunosensors and the immobilization of horseradish peroxidase leading to the direct electron transfer between the redox enzyme and the electrode. The use of catalytic nanoparticles leads to the construction of a reagent‐less immunosensor and the simultaneous detection of DNA and proteins on the same electrode array is demonstrated.

Simultaneous screening for HBV DNA and HCV RNA genomes in blood donations using a novel TaqMan PCR assay

The risk of contracting hepatitis from blood transfusions is estimated to be 1 in 63 000 in the case of hepatitis B virus (HBV) and 1 in 103 000 in the case of hepatitis C virus (HCV). In some countries (Germany, USA and England, for example), molecular protocols are evaluated to detect viral genomes in blood donations in order to reduce the seroconversion period. However, no such method is available currently to screen large series samples for HBV and HCV. While strategies involving the pooling of plasma samples have been proposed and tested in Germany, there is the question of sensitivity. We developed a novel approach to screen for HBV and HCV based on the TaqMan technology that allows for the quantification of an amplified fragment during PCR analysis ( Lee et al., 1993). This approach is more sensitive than other quantification methods. As a first step primers and probes were designed to detect the different sub-types of HBV and HCV genomes. We then optimized the reaction conditions in order to screen for the two viruses at the same time. The observed sensitivity is less than 50 molecules per ml for HBV and less than 50 molecules per ml for HCV. This assay is, to our knowledge, the first that allows the simultaneous detection of DNA and RNA viral genomes. In conclusion, this TaqMan approach could be used as a single test to screen for HBV and HCV genomes in a series of 96 samples in less than 5 h. Such an approach is a first step for development of automation allowing a systematic screening of blood donations.

A simple fluorometric assay for the determination of cell numbers

A sensitive fluorometric assay was developed for the enumeration of cells in microtiter plates. This assay is based on the fluorescence enhancement of propidium iodide (PI) upon binding with double-stranded nucleic acids. This fluorochrome is compatible with a wide range of reagents commonly used in the laboratory, thus washing the cells before staining is not necessary. PI, together with Triton-X 100 and EDTA, was added directly to the cell culture. After 16–18 h incubation at room temperature, intensity of fluorescence was determined with a micro-plate fluorometer. This quick and simple method is sensitive for as little as 1.95 × 10 3 mononuclear leukocytes, and provides a linear correlation ( r=0.999) between cell number and fluorescence up to 1 × 10 6 cells. Since PI has a large Stokes shift with excitation wavelength at common visible range and emission wavelength far out in the red region of the spectrum, it allows simultaneous detection of DNA and other fluorescent compounds such as calcein and fluorescein. This assay may prove to be a valuable alternative for cell number determination.

Simultaneous detection of DNA and RNA by differential polymerase chain reaction (DIFF-PCR).

A new technique, the differential polymerase chain reaction (DIFF-PCR), allows the simultaneous amplification of DNA and homologous RNA in a single assay by the combination of DNA-PCR and RNA-PCR on the same target. DNA-PCR amplifies a selected segment of dsDNA, whereas RNA-PCR amplifies a complementary DNA (cDNA), produced by reverse transcription of RNA. In a mixture of target DNA and RNA, DNA is amplified using a combination of sense and antisense primers under high-stringency conditions giving a D-amplicon. RNA is first reverse-transcribed with a primer carrying a nontarget 5' end into a tagged cDNA at low stringency. Tagged cDNA is subsequently amplified, providing an R-amplicon smaller in size than the D-amplicon. By quantifying the relative amounts of amplified RNA and homologous DNA, a sensitive measure for the transcription rate of a defined DNA segment is obtained. Thus, DIFF-PCR may serve as a useful tool for monitoring gene expression as well as for studying gene regulation and gene function.

Immunocytochemical Detection of DNA Synthesis in Plant Cells

Summary An immunocytochemical protocol has been developed to detect DNA synthesis in plant protoplasts and cells using soybean as a model system. Cells were labelled with the thymidine analog, 5-bromo-2deoxyuridine (BrdU) and fixed for antibody treatments. Formaldehyde fixation gave better results than a mixture of methanol and acetic acid. Suspension cells required an enzyme treatment to partially remove the cell wall. Fixed protoplasts and cells were extracted with Triton X-100 and reacted with a monoclonal antibody against BrdU. The BrdU labelled nuclei were visualized with either enzyme- or fluorescein-conjugated secondary antibodies. Aphidicolin reduced the number of nuclei labelled by more than 50 % at lopg-ml-', inhibited the labelling completely at 501g- ml-', confirming that the labelling was due to replicative nuclear DNA synthesis. Immunocytochemistry is simpler than autoradiography and allows simultaneous detection of DNA and other antigens including, for example, microtubules. This technique should be widely useful in studies of plant DNA replication.