Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by Loop-Mediated Isothermal Amplification (LAMP)

<p><b>Background:</b> The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay.</p> <p><b>Methodology/Principal Findings:</b> For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards.</p> <p><b>Conclusions/Significance:</b> This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.</p&gt

Synthesis, structure, DNA binding studies and nuclease activities of two luminescent neodymium complexes

<p>Two neodymium(III) complexes, [Nd(Phen)(NO₃)₃(DMF)₂] (<b>1</b>) and [Nd(Phen)₂(NO₃)₃] (<b>2</b>) (phen = 1,10-phenanthroline; DMF = dimethylformamide), have been synthesized with a view to design artificial luminescent nucleases and nuclease mimics. The complexes were characterized by spectroscopic, powder, and single crystal XRD studies. The complexes, as expected, have luminescent properties. The DNA binding studies of both complexes have been carried out by spectroscopic studies e.g. electronic absorption (UV–Vis), fluorescence emission as well as viscosity measurements. The nuclease activity of the complexes has been established by gel electrophoresis using pUC19 circular plasmid DNA. The results of DNA binding as well as DNA cleavage activity and the model studies of interaction with <i>p</i>NPP indicate that both neodymium complexes demonstrate nuclease activity through phosphoester bond cleavage.</p

Expression of Trypanosoma congolense antigens in spodoptera frugiperdia insect cells

Transcripts which encode two metacyclic-form-specific variable surface glycoproteins (mVSGs) of Trypanosoma congolense IL3000 have been cloned into baculovirus expression vectors using a novel transfer vector, pAcL11. One of the recombinant baculoviruses (AcVSG1) expressed a mVSG as a glycoprotein with a signal peptide which was cleaved in this expression system, whereas the other one (AcVSG2) expressed an uprocessed protein. From 1 liter of culture containing 109 spodoptera frugiperda cells infected with the recombinant baculoviruses, 10 and 30 mg of mVSG1 and mVSG2, respectively, were obtained. Monospecific polyclonal antibodies produced by immunization of mice with the recombinant proteins reacted specifically with the respective proteins and showed no cross-reactivities between mVSG1 and mVSG2 in imunoblot assays. The antibodies to each of the proteins stained only the surface of a proportion of intact fixed T. congolense IL3000 metacyclic forms. It was possible to determine from these studies that, on the average, the parasites expressing mVSG1 constitute approximately 45 percent of the metacyclic population of T. congolense IL3000 maintained in in vitro cultures, whereas those that express mVSG2 constitute approximately 20 percent