Development, validation and evaluation of a rapid PCR-nucleic acid lateral flow immuno-assay for the detection of Plasmodium and the differentiation between Plasmodium falciparum and Plasmodium vivax.

BACKGROUND: Molecular tools are very sensitive and specific and could be an alternative for the diagnosis of malaria. The complexity and need for expensive equipment may hamper implementation and, therefore, simplifications to current protocols are warranted. METHODS: A PCR detecting the different Plasmodium species and differentiating between Plasmodium falciparum and Plasmodium vivax was developed and combined with a nucleic acid lateral flow immuno-assay (PCR-NALFIA) for amplicon detection. The assay was thoroughly evaluated for the analytical sensitivity and specificity in the laboratory, the robustness and reproducibility in a ring trial and accuracy and predictive value in a field trial. RESULTS: The analytical sensitivity and specificity were 0.978 (95% CI: 0.932-0.994) and 0.980 (95% CI: 0.924-0.997), respectively, and were slightly less sensitive for the detection of P. vivax than for P. falciparum. The reproducibility tested in three laboratories was very good (k = 0.83). This evaluation showed that the PCR machine used could influence the results. Accuracy was evaluated in Thailand and compared to expert microscopy and rapid diagnostic tests (RDTs). The overall and P. falciparum-specific sensitivity and specificity was good ranging from 0.86-1 and 0.95-0.98 respectively, compared to microscopy. Plasmodium vivax detection was better than the sensitivity of RDT, but slightly less than microscopy performed in this study. CONCLUSION: PCR-NALFIA is a sensitive, specific and robust assay able to identify Plasmodium species with good accuracy. Extensive testing including a ring trial can identify possible bottlenecks before implementation and is therefore essential to perform in additon to other evaluations.

Carbon nanoparticles as detection labels in antibody microarrays. Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli.

The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1, vt2, eae, and ehxA) and the gene for E. coli 16S (hui). Optimization was performed using a Box-Behnken design, and the limit of detection for each virulence factor was established. Finally, this NAMIA using CNPs was tested with DNA from 48 field strains originating from cattle feces, and its performance was evaluated by comparing results with those achieved by the reference method q-PCR. All factors tested gave sensitivity and specificity values higher than 0.80 and efficiency values higher than 0.92. Kappa coefficients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used for vt1, eae, and ehxA, and a perfect agreement (k = 1) for vt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed fit for purpose, i.e., it is valuable for fast screening of amplified genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays.

Secreted production of collagen-inspired gel-forming polymers with high thermal stability in Pichia pastoris.

Previously, we have shown that gel-forming triblock proteins, consisting of random coil middle blocks and trimer-forming (Pro-Gly-Pro)(9) end blocks, are efficiently produced and secreted by the yeast Pichia pastoris. These end blocks had a melting temperature (T(m)) of ∼41°C (at 1.1 mM of protein). The present work reveals that an increase of T(m) to ∼74°C, obtained by extension of the end blocks to (Pro-Gly-Pro)(16), resulted in a five times lower yield and partial endoproteolytic degradation of the protein. A possible cause could be that the higher thermostability of the longer (Pro-Gly-Pro)(16) trimers leads to a higher incidence of trimers in the cell, and that this disturbs secretion of the protein. Alternatively, the increased length of the proline-rich (Pro-Gly-Pro)(n) domain may negatively influence ribosomal translation, or may result in, for example, hydrophobic aggregation or membrane-active behavior owing to the greater number of closely placed proline residues. To discriminate between these possibilities, we studied the production of molecules with randomized end blocks that are unable to form triple helices. The codon- and amino acid composition of the genes and proteins, respectively, remained unchanged. As these nontrimerizing molecules were secreted intact and at high yield, we conclude that the impaired secretion and partial degradation of the triblock with (Pro-Gly-Pro)(16) end blocks was triggered by the occurrence of intracellular triple helices. This degradation was overcome by using a yapsin 1 protease disruptant, and the intact secreted polymer was capable of forming self-supporting gels of high thermal stability.

Secreted production of self-assembling peptides in Pichia pastoris by fusion to an artificial highly hydrophilic protein.

The undecapeptides CH(3)CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH(2) (P(11)-2) and CH(3)CO-Gln-Gln-Orn-Phe-Orn-Trp-Orn-Phe-Orn-Gln-Gln-NH(2) (P(11)-14) have unique self-assembly characteristics and broad application potential. Originally, these peptides were produced by chemical synthesis, which is costly and difficult to scale up to industrial levels in an economically feasible way. This article describes the efficient secreted production of these peptides (with free termini and ornithines replaced with lysines) in the methylotrophic yeast Pichia pastoris. The peptides were produced as enterokinase-cleavable fusions to the C-terminus of an artificial Solubility-Enhancing Protein (SEP). In vitro, the fused highly hydrophilic SEP proved to prevent self-assembly of the peptides. The SEP domain also facilitates product detection and allows convenient separation of the fusion protein from the broth by simple salt precipitation. After cleavage of the purified fusion protein with enterokinase, the free undecapeptides were obtained and P(11)-2 spontaneously assembled into a self-supporting gel, as intended. The properties of the SEP carrier could be advantageous for the production of other peptides.

Precision gels from collagen-inspired triblock copolymers.

Gelatin hydrogels find broad medical application. The current materials, however, are from animal sources, and their molecular structure and thermal properties cannot be controlled. This study describes recombinant gelatin-like polymers with a general design that inherently offers independent tuning of the cross-link density, melting temperature, and biocompatibility of the gel. The polymers contain small blocks with thermoreversible trimerization capacity and defined melting temperature, separated by hydrophilic nontrimerizing blocks defining the distance between the knot-forming domains. As an example, we report the secreted production in yeast at several g/L of two nonhydroxylated approximately 42 kDa triblock copolymers with terminal trimerizing blocks. Because only the end blocks formed cross-links, the molecular architecture of the gels is much more defined than that of traditional gelatins. The novel hydrogels had a approximately 37 degrees C melting temperature, and the dynamic elasticity was independent of the thermal history. The concept allows to produce custom-made precision gels for biomedical applications.

Biosynthesis of an amphiphilic silk-like polymer.

An amphiphilic silk-like protein polymer was efficiently produced in the yeast Pichia pastoris. The secreted product was fully intact and was purified by solubilization in formic acid and subsequent precipitation of denatured host proteins upon dilution with water. In aqueous alkaline solution, the negatively charged acidic polymer assumed extended helical (silk III-like) and unordered conformations. Upon subsequent drying, it assumed a conformation rich in beta-turns. In water at low pH, the uncharged polymer aggregated and the solution became turbid. Concentrated solutions in 70% (v/v) formic acid slowly formed gels. Replacement of the formic acid-water mixture with methanol and subsequent drying resulted in beta-sheets, which stacked into fibril-like structures. The novel polymer instantaneously lowered the air-water interfacial tension under neutral to alkaline conditions and reversed the polarity of hydrophobic and hydrophilic solid surfaces upon adsorption.

Overproduction of heterologous mannitol 1-phosphatase: a key factor for engineering mannitol production by Lactococcus lactis.

To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.

Characterisation of Aspergillus niger prolyl aminopeptidase.

We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity. The overexpressed enzyme was subsequently purified and characterised. The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides. It is the first enzyme of its kind from a eukaryotic organism that has been characterised.