Homogeneous and digital proximity ligation assays for the detection of Clostridium difficile toxins A and B.

BACKGROUND: The proximity ligation assay (PLA) detects proteins via their interaction with pairs of proximity probes, which are antibodies coupled to noncomplementary DNA oligonucleotides. The binding of both proximity probes to their epitopes on the target protein brings the oligonucleotides together, allowing them to be bridged by a third oligonucleotide with complementarity to the other two. This enables their ligation and the detection of the resulting amplicon by real-time quantitative PCR (qPCR), which acts as a surrogate marker for the protein of interest. Hence PLA has potential as a clinically relevant diagnostic tool for the detection of pathogens where nucleic acid based tests are inconclusive proof of infection. METHODS: We prepared monoclonal and polyclonal proximity probes targeting Clostridium difficile toxins A (TcdA) and B (TcdB) and used hydrolysis probe-based qPCR and digital PCR (dPCR) assays to detect antibody/antigen interactions. RESULTS: The performance of the PLA assays was antibody-dependent but both TcdA and TcdB assays were more sensitive than comparable ELISAs in either single- or dualplex formats. Both PLAs could be performed using single monoclonal antibodies coupled to different oligonucleotides. Finally, we used dPCR to demonstrate its potential for accurate and reliable quantification of TcdA. CONCLUSIONS: PLA with either qPCR or dPCR readout have potential as new diagnostic applications for the detection of pathogens where nucleic acid based tests do not indicate viability or expression of toxins. Importantly, since it is not always necessary to use two different antibodies, the pool of potential antibodies useful for PLA diagnostic assays is usefully enhanced.

Stem-loop RT-qPCR for microRNA expression profiling.

Quantification of the microRNAs (miRNAs) in cells or tissues is a crucial step in understanding their biological functions. Development of the stem-loop reverse transcription procedure and TaqMan(®) miRNA assays enables accurate detection of miRNA expression levels by quantitative PCR. Increased experimental throughput permits the expression screening of larger number of miRNAs with small amounts of sample. Here, we demonstrate the use of both TaqMan(®) Array Card and OpenArray(®) platforms to accurately determine the level of miRNA gene expression in biological samples.

Enrichment of sequencing targets from the human genome by solution hybridization.

To exploit fully the potential of current sequencing technologies for population-based studies, one must enrich for loci from the human genome. Here we evaluate the hybridization-based approach by using oligonucleotide capture probes in solution to enrich for approximately 3.9 Mb of sequence target. We demonstrate that the tiling probe frequency is important for generating sequence data with high uniform coverage of targets. We obtained 93% sensitivity to detect SNPs, with a calling accuracy greater than 99%.

Planetary-scale terrain composition.

Many interrelated planetary height map and surface image map data sets exist, and more data are collected each day. Broad communities of scientists require tools to compose these data interactively and explore them via real-time visualization. While related, these data sets are often unregistered with one another, having different projection, resolution, format, and type. We present a GPU-centric approach to the real-time composition and display of unregistered-but-related planetary-scale data. This approach employs a GPGPU process to tessellate spherical height fields. It uses a render-to-vertex-buffer technique to operate upon polygonal surface meshes in image space, allowing geometry processes to be expressed in terms of image processing. With height and surface map data processing unified in this fashion, a number of powerful composition operations may be uniformly applied to both. Examples include adaptation to nonuniform sampling due to projection, seamless blending of data of disparate resolution or transformation regardless of boundary, and the smooth interpolation of levels of detail in both geometry and imagery. Issues of scalability and precision are addressed, giving out-of-core access to giga-pixel data sources, and correct rendering at scales approaching one meter.