Lab-on-a-Film disposable for genotyping multidrug-resistant Mycobacterium tuberculosis from sputum extracts.

We describe a Lab-on-a-Film disposable that detects multidrug-resistant tuberculosis (MDR-TB) from sputum extracts. The Lab-on-a-Film disposable consists of 203 gel elements that include DNA sequences (probes) for 37 mutations, deletions, or insertion elements across 5 genes (including an internal control). These gel elements are printed on a flexible film, which costs approximately 500 times less than microarray glass.

Genotyping Multidrug-Resistant Mycobacterium tuberculosis from Primary Sputum and Decontaminated Sediment with an Integrated Microfluidic Amplification Microarray Test.

There is a growing awareness that molecular diagnostics for detect-to-treat applications will soon need a highly multiplexed mutation detection and identification capability. In this study, we converted an open-amplicon microarray hybridization test for multidrug-resistant (MDR) into an entirely closed-amplicon consumable (an amplification microarray) and evaluated its performance with matched sputum and sediment extracts. Reproducible genotyping (the limit of detection) was achieved with ∼25 genomes (100 fg of DNA) per reaction; the estimated shelf life of the test was at least 18 months when it was stored at 4°C.

Development and clinical testing of a simple, low-density gel element array for influenza identification, subtyping, and H275Y detection.

The objectives of this study were to develop a user-friendly, gel element microarray test for influenza virus detection, subtyping, and neuraminidase inhibitor resistance detection, assess the performance characteristics of the assay, and perform a clinical evaluation on retrospective nasopharyngeal swab specimens. A streamlined microarray workflow enabled a single user to run up to 24 tests in an 8h shift. The most sensitive components of the test were the primers and probes targeting the A/H1 pdm09 HA gene with an analytical limit of detection (LoD) <100 gene copies (gc) per reaction.

Automated, simple, and efficient influenza RNA extraction from clinical respiratory swabs using TruTip and epMotion.

Background: Rapid, simple and efficient influenza RNA purification from clinical samples is essential for sensitive molecular detection of influenza infection. Automation of the TruTip extraction method can increase sample throughput while maintaining performance.

Objectives: To automate TruTip influenza RNA extraction using an Eppendorf epMotion robotic liquid handler, and to compare its performance to the bioMerieux easyMAG and Qiagen QIAcube instruments.

Profiling in situ microbial community structure with an amplification microarray.

The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO(3)(-)) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures.

Rapid, simple influenza RNA extraction from nasopharyngeal samples.

This report describes the development and pre-clinical testing of a new, random-access RNA sample preparation system (TruTip) for nasopharyngeal samples. The system is based on a monolithic, porous nucleic acid binding matrix embedded within an aerosol-resistant pipette tip and can be operated with single or multi-channel pipettors. Equivalent extraction efficiencies were obtained between automated QIAcube and manual TruTip methods at 10(6) gene copies influenza A per mL nasopharyngeal aspirate.

Integrated Amplification Microarrays for Infectious Disease Diagnostics.

This overview describes microarray-based tests that combine solution-phase amplification chemistry and microarray hybridization within a single microfluidic chamber. The integrated biochemical approach improves microarray workflow for diagnostic applications by reducing the number of steps and minimizing the potential for sample or amplicon cross-contamination. Examples described herein illustrate a basic, integrated approach for DNA and RNA genomes, and a simple consumable architecture for incorporating wash steps while retaining an entirely closed system.

Monitoring microbial community structure and dynamics during in situ U(VI) bioremediation with a field-portable microarray analysis system.

The objective of this study was to develop and validate a simple, field-portable, microarray system for monitoring microbial community structure and dynamics in groundwater and subsurface environments, using samples representing site status before acetate injection, during Fe-reduction, in the transition from Fe- to SO(4)(2-)-reduction, and into the SO(4)(2-)-reduction phase. Limits of detection for the array are approximately 10(2)-10(3) cell equivalents of DNA per reaction. Sample-to-answer results for the field deployment were obtained in 4 h.

Cyclin E and survival in patients with breast cancer.

Background: Cyclin E, a regulator of the cell cycle, affects the behavior of breast-cancer cells. We investigated whether levels of cyclin E in the tumor correlated with survival among patients with breast cancer.

Methods: Tumor tissue from 395 patients with breast cancer was assayed for cyclin E, cyclin D1, cyclin D3, and the HER-2/neu oncogene with the use of Western blot analysis.