Oligonucleotide Based Magnetic Bead Capture of Onchocerca volvulus DNA for PCR Pool Screening of Vector Black Flies

The absence of infective larvae of Onchocerca volvulus in the black fly vector of this parasite is a major criterion used to certify that transmission has been eliminated in a focus. This process requires screening large numbers of flies. Currently, this is accomplished by screening pools of flies using a PCR-based assay. The number of flies that may be included in each pool is currently limited by the DNA purification process to 50 flies for Latin American vectors and 100 flies for African vectors. Here, we describe a new method for DNA purification that relies upon a specific oligonucleotide to capture and immobilize the parasite DNA on a magnetic bead. This method permits the reliable detection of a single infective larva of O. volvulus in pools containing up to 200 individual flies. The method described here will dramatically improve the efficiency of pool screening of vector black flies, making the process of elimination certification easier and less expensive to implement

Particle flow assays for fluorescent protein microarray applications

Microarray technology has brought a paradigmatic change in bioanalytics. However, highly sensitive and accurate assays are still needed for a real breakthrough. We present a simple and generic approach for fluorescent signal amplification with fluorescent microparticle labels. The assay principle was demonstrated using a reverse array model consisting of spots of bovine serum albumin with a small fraction of the proteins biotinylated. Specific binding of streptavidin coated fluorescent microparticles to the spots was promoted by applying a controlled continuous microparticle flow. The surface bound beads were visualized and quantified with confocal microscopy images. Comparison with standard fluorescent and flow discrimination assays has revealed several advantages of our approach. First, non-specific particle binding could be reduced to less than 1particle/spot making therefore the visualization of single biomolecular bonds possible. Second, the amplification scheme presented here is generic and can be applied to any fluorescent microarray. Furthermore, this assay makes use of a biotin-streptavidin linkage and can therefore be applied to all kind of assays. Finally, single fluorescent microbeads can be easily visualized with standard optical equipments, so that no high performance equipment is required

Insulin modulates the frequency of Ca2+ oscillations in mouse pancreatic islets

Pancreatic islets can adapt to oscillatory glucose to produce synchronous insulin pulses. Can islets adapt to other oscillatory stimuli, specifically insulin? To answer this question, we stimulated islets with pulses of exogenous insulin and measured their Ca2+ oscillations. We observed that sufficiently high insulin (> 500 nM) with an optimal pulse period (~ 4 min) could make islets to produce synchronous Ca2+ oscillations. Glucose and insulin, which are key stimulatory factors of islets, modulate islet Ca2+ oscillations differently. Glucose increases the active-to-silent ratio of phases, whereas insulin increases the period of the oscillation. To examine the dual modulation, we adopted a phase oscillator model that incorporated the phase and frequency modulations. This mathematical model showed that out-of-phase oscillations of glucose and insulin were more effective at synchronizing islet Ca2+ oscillations than in-phase stimuli. This finding suggests that a phase shift in glucose and insulin oscillations can enhance inter-islet synchronization