Detection Of Specific Targets Research Articles

Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay

Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5' and 3' ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 10(4). A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.

INFECTION WITH CHLAMYDIA PNEUMONIAE INDUCES A PRONOUNCED PRO-INFLAMMATORY PHENOTYPE IN HUVEC VIA INDUCTION OF EGR-1 AND NFAT

The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA MasterPLUS HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Real-time PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs.

Effective Cell Identification and Segmentation in Fluorescence Microscopy with New Fluorescent Whole Cell Stains

Abstract High-content screening (HCS) and other quantitative cellular imaging assay methods that use fluorescence microscopy require effective fluorescent labeling and identification of the target cell(s). Fluorescent probes that stain cells as the primary objects are used to identify and count individual cells, as well as to define the region(s) of each cell to which target-specific image analysis is applied. For this purpose, the primary object can be a major component of the cell, such as the nucleus or a large organelle, or the whole cell itself. When the whole cell is the primary object, a high-quality cell stain is needed to delineate the intact cell from bordering cells without also interfering in target-specific detection and analysis. Thermo Scientific Cellomics ® Whole Cell Stains (Thermo Fisher Scientific Inc., Rockford, Ill.), available in blue, green, orange and red, provides image staining and the ability to quantify the whole cell volume in HCS and fluorescence microscopy assays.

Impact of consumption of probiotic lactobacilli-containing yogurt on microbial composition in human feces

An in vivo study was carried out to determine the effect of consuming probiotic lactobacilli-containing yogurt on the composition of microbiota in the human gut. Fifteen healthy adults ingested a daily serving of one of three commercial yogurts (two of the products contained a probiotic lactobacilli strain) for 20 days. Fecal samples at defined time points before, during, and after the period of yogurt ingestion were collected and analyzed. The fecal population of lactobacilli was determined by a culture-based method and subsequent colony PCR for the identification of species. Six predominant bacterial groups in the fecal samples were quantitatively determined based on a sequence-specific SSU rRNA cleavage method coupled with a suite of oligonucleotide probes, which was optimized for the target-specific detection of bacterial groups inhabiting human feces. In the ingestion period, one probiotic strain was detected in the feces of all five subjects who consumed the yogurt containing the strain, while the other strain was detected in three of another five subjects. The population levels of the two major groups ( Bacteroides and Prevotella, and the Clostridium coccoides– Eubacterium rectale group) in the fecal samples tended to change in response to the ingestion but the change did not seem to be dependent on the product-specific property of each yogurt. These results suggest that the human fecal bacterial community could be altered by ingesting yogurt, although whether probiotic lactobacilli are present or absent in the yogurt does not seem to be a factor in this change.

Type classification, color estimation, and specific target detection of moving targets on public streets

This paper describes a vision system that recognizes moving targets such as vehicles and pedestrians on public streets. This system can: (1) classify targets {vehicle, pedestrian, others} and, for "vehicles," discriminate vehicle types and (2) estimate the main colors of targets. According to the input images to the system, the categories of targets are set as {mule (golf cart for workers), sedan, van, truck, pedestrian (single or plural), and other (such as noise)}. Their colors are set as six color groups {red, orange, yellow; green; blue, light blue; white, silver, gray; dark blue, dark gray, black; dark red, dark orange}. In this experiment, we collected images of targets from 9: 00 a.m. to 5: 00 p.m. on sunny and cloudy days as system training samples. The recognition ratio was 91.1% under the condition that both the recognition results of type and color agreed with the operator's judgment. In addition, the system can detect predefined specific targets such as delivery vans, post office vans, and police cars by combining recognition results for type and color. The recognition ratio for specific targets was 92.9%. For the classification and estimation of targets, we employed a statistical linear discrimination method (linear discriminant analysis, LDA) and a nonlinear decision rule (weighted K-nearest neighbor rule, K-NN).

Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis.

Multiplex PCR procedures were developed for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready), maize (event 176, Bt11, Mon810, T14/25), and canola (GT73, HCN92/28, MS8/RF3, Oxy 235). Internal control targets (invertase gene in corn, lectin and beta-actin genes in soybean, and cruciferin gene in canola) were included as appropriate to assess the efficiency of all reactions, thereby eliminating any false negatives. Primer combinations that allowed the identification of specific lines were used. In one system of identification, simultaneous amplification profiling (SAP), rather than target specific detection, was used for the identification of four GM maize lines. SAP is simple and has the potential to identify both approved and nonapproved GM lines. The template concentration was identified as a critical factor affecting efficient multiplex PCRs. In canola, 75 ng of DNA template was more effective than 50 ng of DNA for the simultaneous amplification of all targets in a reaction volume of 25 microL. Reliable identification of GM canola was achieved at a DNA concentration of 3 ng/microL, and at 0.1% for GM soybean, indicating high levels of sensitivity. Nonspecific amplification was utilized in this study as a tool for specific and reliable identification of one line of GM maize. The primer cry1A 4-3' (antisense primer) recognizes two sites on the DNA template extracted from GM transgenic maize containing event 176 (European corn borer resistant), resulting in the amplification of products of 152 bp (expected) and 485 bp (unexpected). The latter fragment was sequenced and confirmed to be Cry1A specific. The systems described herein represent simple, accurate, and sensitive GMO detection methods in which only one reaction is necessary to detect multiple GM target sequences that can be reliably used for the identification of specific lines of GMOs.

Multiplexed, Targeted Gene Expression Profiling and Genetic Analysis on Electronic Microarrays

Electronic microarrays comprise independent microelectrode test sites that can be electronically biased positive or negative, or left neutral, to move and concentrate charged molecules such as DNA and RNA to one or more test sites. We developed a protocol for multiplexed gene expression profiling of mRNA targets that uses electronic field-facilitated hybridization on electronic microarrays. A multiplexed, T7 RNA polymerase-mediated amplification method was used for expression profiling of target mRNAs from total cellular RNA; targets were detected by hybridization to sequence-specific capture oligonucleotides on electronic microarrays. Activation of individual test sites on the electronic microarray was used to target hybridization to designated subsets of sites and allow comparisons of target concentrations in different samples. We used multiplexed amplification and electronic field-facilitated hybridization to analyze expression of a model set of 10 target genes in the U937 cell line during lipopolysaccharide-mediated differentiation. Performance of multiple genetic analyses (single-nucleotide polymorphism detection, gene expression profiling, and splicing isoform detection) on a single electronic microarray was demonstrated using the ApoE and ApoER2 genes as a model system. Targets were detected after a 2-min hybridization reaction. With noncomplementary capture probes, no signal was detectable. Twofold changes in target concentration were detectable throughout the ( approximately 64-fold) range of concentrations tested. Levels of 10 targets were analyzed side by side across seven time points. By confining electronic activation to subsets of test sites, polymorphism detection, expression profiling, and splicing isoform analysis were performed on a single electronic microarray. Microelectronic array technology provides specific target detection and quantification with advantages over currently available methodologies for targeted gene expression profiling and combinatorial genomics testing.

In situ amplification using universal energy transfer-labeled primers.

We developed an amplification detection system in which a universal energy transfer-labeled primer (UniPrimer) is used in combination with any target-specific primer pair. The target specific primers each have a 5' tail sequence, which is homologous to the 3' end of the UniPrimer which, in turn, has a hairpin structure on the 5' end. The hairpin structure brings the fluorophore and quencher into close proximity when the primer is free in solution, providing efficient quenching. When the primer is incorporated into the PCR product, the hairpin structure is unfolded and a fluorescent signal can be detected. Using hepatitis C and human papillomavirus as model systems, this study demonstrates several advantages in the hot-start in situ PCR technique with the UniPrimer system, including target specific detection of one DNA copy per cell without a separate in situ hybridization step and detection of an RNA target by RT in situ PCR without overnight DNase digestion. The UniPrimer-based in situ PCR allows rapid and simple detection of any DNA or RNA target without concern for the background from DNA repair invariably evident in paraffin-embedded tissue when a labeled nucleotide is used.

Imaging Q-switched CO_2 laser radar with heterodyne detection: design and evaluation

A Q-switched CO2 laser radar with heterodyne detection has been designed and evaluated. A simplified theory has been used to optimize the Q-switched laser for high-resolution ranging. The return signal statistics from diffuse, glint, and topographical targets have been investigated, and statistical distributions have been fitted to the experimental data. Detection of specific targets in laser radar images using range gating has also been studied.