Electrochemical DNA hybridization detection using peptide nucleic acids and [Ru(NH3)6]3+ on gold electrodes.

An electrochemical DNA hybridization detection method based on the electrostatic interactions of [Ru(NH3)6]3+ cations with the anionic phosphate backbone of DNA is proposed. PNA molecules are immobilized as capture probes on the gold substrate. The cationic ruthenium complexes do not interact electrostatically with the PNA probes due to the absence of the anionic phosphate groups on the PNA probes. But after hybridization, [Ru(NH3)6]3+ is adsorbed on the DNA backbone, giving a clear hybridization detection signal in ac voltammetry. The analytical parameters (sensitivity, selectivity and reproducibility) are evaluated. Very good discrimination against the single-base mismatch A2143G, internal to the 23S rRNA gene of Helicobacter pylori, is observed. Moreover the system is successfully applied to the detection of complementary PCR amplicons.

Colorimetric solid-phase capture hybridization assay for detection of amplified Borrelia burgdorferi DNA.

To simplify detection procedures of DNA fragments resulting from PCR, we developed a colorimetric microplate hybridization assay. This format was used for the identification of Borrelia burgdorferi sensu lato, the causal agent of Lyme disease. The system relied on the use of a specific capture probe covalently linked to polystyrene plates and a specific polybiotinylated detection probe. DNA fragments, resulting from PCR and sandwiched between these two probes, were detected by enzymatic color development. The new detection format outperformed agarose gel electrophoresis of PCR products in sensitivity and specificity Moreover, in view of its rapidity and simplicity, the system proved appropriate for the routine diagnostic analysis of clinical specimens from Lyme disease patients. The proposed detection format can be adapted easily to other DNA targets and is suitable for automation.

Protein kinase activity in Helicobacter pylori.

Based on the predictive analysis of the cellular protein content from the complete genome sequence of Helicobacter pylori, discrepant results were previously reported concerning the occurrence of a protein kinase in this bacterium. To solve this ambiguity, we have directly assayed cellular extracts for their capacity of phosphorylating endogenous proteins. At least eight different proteins, ranging from 24 to 200 kDa, were found to be phosphorylated to a varying extent. Individual measurement of their phosphoamino acid composition showed that they all were modified at serine residues. These data indicate that H. pylori does contain a protein-serine kinase activity.

A PCR based DNA hybridisation capture system for the detection of human cytomegalovirus. A comparative study with other identification methods.

A simple, sensitive and specific colourimetric hybridisation method for the detection of HCMV DNA in clinical specimens is described. This method combines a PCR assay with a sensitive sandwich hybridisation assay. It relies on the use of a specific capture probe linked covalently to polystyrene microplates and a specific polybiotinylated detection probe. Amplified DNA fragments, sandwiched between these two probes, are detected by an enzymatic colour reaction. This PCR-based colourimetric hybridisation method was compared with other known HCMV detection methods. Clinical specimens (n = 145, corresponding to 106 patients) were tested by both a nested PCR assay and this colourimetric hybridisation method; and by either the culture method or the pp65 antigenaemia test depending on the type of sample used. The results showed that the PCR-based hybridisation method has a specificity similar to tissue culture, known as the conventional gold standard method, and could be used for the examination of the clinical specimens.

Detection and identification of human papilloma viral DNA, types 16, 18, and 33, by a combination of polymerase chain reaction and a colorimetric solid phase capture hybridisation assay.

A colorimetric microplate hybridization assay was developed previously to simplify detection procedures of DNA fragments resulting from polymerase chain reactions (PCR). This format has now been adapted for the simultaneous detection and identification of three human papillomavirus (HPV), types 16, 18 and 33, associated frequently with cervical cancer. This post-PCR detection system uses three type-specific capture oligonucleotides linked covalently to a single microplate well and three type-specific multibiotinylated oligonucleotidic probes for detection. It therefore offers a double specificity; the first is conferred by pairs of primers, specific of each type of virus tested, and the second, by the sets of capture and detection probes which are complementary to internal regions of the amplified DNA fragments. The detection format outperformed agarose gel electrophoresis of amplified DNA products in sensitivity and specificity. The rapidity and simplicity of this hybridisation system would justify its use in routine diagnostic examination of cervical specimens (smears and biopsies).

PCR assay fails to detect molluscum contagiosum virus-related sequences in AIDS-related Kaposi's sarcoma.

Previous PCR-based studies have demonstrated the presence of various viral DNA or RNA sequences in Kaposi's sarcoma (KS) tissues. To date, only human herpesvirus 8 (HHV-8) DNA sequences are found consistently in KS. The putative role of this agent in KS pathogenesis remains, however, to be determined; HHV-8 could infect populations endemically and could be reactivated in patients with KS. A close association between AIDS-related KS and molluscum contagiosum occurrence was found and this study was conducted primarily to search for the presence of molluscum contagiosum virus DNA sequences in KS. Frozen KS samples were examined for the presence of both HHV-8 and molluscum contagiosum virus DNA sequences by PCR. Despite a high rate of co-infection, no molluscum contagiosum virus (MCV) DNA sequence could be found in the KS samples whereas HHV-8 was uniformly detected. These results suggest that the high prevalence of MCV in AIDS patients with KS relies on a mode of transmission common for HHV-8 and molluscum contagiosum virus rather than on a multiviral etiology of KS. They may also indicate a particular susceptibility of the host to viral reactivation. If this is so, the failure to detect MCV DNA sequences in KS tissues by PCR indicates that locally produced or released cyotokines are not involved in the latter process.

Assignment of Borrelia burgdorferi strains G25 and VS461 to the Borrelia garinii and Borrelia afzelii genospecies, respectively: a comparison of OspA protein sequences.

The nucleotide sequence encoding the Outer Surface Protein A (OspA) from two Borrelia burgdorferi sensu lato isolates, G25 and VS461, has been determined. On the basis of a phylogenetic analysis, strains G25 and VS461 were respectively assigned to the B. garinii and B. afzelii genospecies. Comparative analysis of OspA proteins from 26 different B. burgdorferi sensu lato strains involved in Lyme disease indicated a higher heterogeneity in the B. garinii genospecies than in the two other genospecies, B. burgdorferi sensu stricto and B. afzelii.

Identification of an elicitin gene cluster in Phytophthora cinnamomi.

Elicitins are a group of highly conserved proteins secreted by species of Phytophthora and a species of the related genus Pythium, Pythium vexans. Some of these proteins act as inducers of the necrotic hypersensitive-like response and the associated systemic acquired resistance phenomenon, in some species. We cloned and characterised the cinnamomin-beta and -alpha genes and two related elicitin genes from Phytophthora cinnamomi. These four open reading frames (ORFs) are clustered in tandem pairs. Two out of these four genes present homologies with the basic and acidic elicitin groups; but the two others encode, if expressed, elicitin isoforms exhibiting homologies with the class II of highly acidic elicitins.

Transmission cycles of Borrelia burgdorferi sensu lato involving Ixodes ricinus and/or I. hexagonus ticks and the European hedgehog, Erinaceus europaeus, in suburban and urban areas in Switzerland.

The European hedgehog, Erinaceus europaeus Linnaeus, 1758, is a common host of Ixodes ricinus L. and I. hexagonus Leach, vectors of the Lyme disease spirochaete, Borrelia burgdorferi sensu lato. To investigate whether hedgehogs are reservoirs for B. burgdorferi, hedgehogs were captured in a suburban area suitable for both tick species and in an urban area where I. ricinus is absent. The infection status of the hedgehogs was determined by xenodiagnosis using I. ricinus and I. hexagonus larvae. I. hexagonus and/or I. ricinus were found on all hedgehogs (n = 8) from the suburban area. In contrast, only I. hexagonus was infesting animals (n = 5) from the urban area. A total of 12/13 hedgehogs harboured B. burgdorferi infected ticks. Xenodiagnostic I. ricinus and I. hexagonus larvae that fed on hedgehogs became infected. The results clearly show that European hedgehogs are reservoir hosts of the Lyme disease spirochetes. DNA of B. burgdorferi sensu stricto, B. garinii and B. afzelii was detected in culture from ear biopsy and needle aspiration material and characterized by using a genospecies-specific PCR assay. One hedgehog presented a mixed infection of the skin with B. burgdorferi sensu stricto and B. garinii. This study also identifies an enzootic transmission cycle in an urban area involving E. europaeus and I. hexagonus. The close association of I. hexagonus with the burrows of its hosts mean that the risks of contact between I. hexagonus and humans may be low.

Protective immunity against Ixodes ricinus induced by a salivary serpin.

Iris is a specific elastase inhibitor expressed in the salivary glands of the hard tick Ixodes ricinus. It belongs to the superfamily of serpins and interferes with both haemostasis and the immune response of the host. In this study, we first show that Iris is expressed in nymphs but not in the female midgut nor in males. We also show that Iris is present in the saliva. To examine its potency as anti-tick vaccine candidate, we set up three models of I. ricinus infestation on immunized animals: nymphs on mice, and adults and nymphs on rabbits. We report the rise of neutralizing antibodies following immunization of rabbits and mice. This comes with a significant protective immunity against ticks in rabbits only, resulting in a 30% mortality rate and a diminution of weight gain in both nymphs and adults and a prolongation of blood feeding time in adults. This is the first report on an anti-tick vaccine trial on I. ricinus using a protein able to interact with both host immunity and haemostasis, as a vaccinating antigen.

Ixodes ticks belonging to the Ixodes ricinus complex encode a family of anticomplement proteins.

The alternative pathway of complement is an important innate defence against pathogens including ticks. This component of the immune system has selected for pathogens that have evolved countermeasures. Recently, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., 2000; J. Biol. Chem. 275, 18717-18723). Here, we isolated two different sequences, similar to Isac, from the transcriptome of I. ricinus salivary glands. Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins, called I. ricinus anticomplement (IRAC) protein I and II, are coexpressed constitutively in I. ricinus salivary glands and are upregulated during blood feeding. Also, we demonstrated that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses demonstrate that ticks belonging to the Ixodes ricinus complex encode a family of relatively small anticomplement molecules undergoing diversification by positive Darwinian selection.

Complexity of the major surface protease (msp) gene organization in Leishmania (Viannia) braziliensis: evolutionary and functional implications.

The major surface protease (msp or gp63) of Leishmania plays a major role in the host-parasite interaction. We analysed here the structure of the msp gene locus in Leishmania (Viannia) braziliensis and compared it to results obtained in other species. Physical mapping of cosmid contigs revealed a minimum of 37 genes per haploid genome and at least 8 different msp gene families. Within the same organism, these genes showed a nucleotide sequence varying in certain stretches from 3 to 34%, and a mosaic structure. From an evolutionary point of view, major differences were observed between subgenera Viannia and Leishmania, both in terms of msp gene number and sequence. Within subgenus Viannia, phenetic analysis revealed three clusters in which sequence variants of L. (Viannia) braziliensis and L. (Viannia) guyanensis were interspersed. Functional implications of our results were explored from predicted L. (Viannia) braziliensis protein sequences: regions encoding the msp catalytic site showed a conserved sequence, while regions encoding surface domains possibly involved in the host-parasite interaction (macrophage adhesion sites and immunodominant B-cell and T-cell epitopes) were variable. We speculate that this would be an adaptive strategy of the parasite.

Glycosylation of the amyloid peptide precursor containing the Kunitz protease inhibitor domain improves the inhibition of trypsin.

The amyloid beta peptide (A beta P) is the major constituent of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease. This peptide is obtained from transmembrane amyloid protein precursors (APP) which sometimes contain a Kunitz protease inhibitor (KPI) insert in their extracellular domain and therefore are able to inhibit serine proteases. Expression of the transmembrane and the secreted APP containing the KPI domain was obtained by transient transfection of COS-1 cells. The overexpressed proteins were detected in immunoblotting experiments and inhibition of trypsin was analyzed using reverse enzymography. Our results indicate that post-translational modifications including glycosylation improve the inhibition of trypsin by the APP containing the KPI domain.

Automatic synthesis of multifork-like structures for multiple labeling of oligonucleotides: application to diagnosis of Lyme disease.

Multifork-like structures have been designed for multiple labelling of oligonucleotides. They rely on the synthesis of new non nucleosidic phosphoramidites which can be used in automatic DNA synthesis. The new compounds allowed tagging of any oligonucleotide and provided reactive primary amino groups for non isotopic labelling. Polybiotinylated oligonucleotides have been tested in hybridization assays using a corresponding complementary sequence covalently linked to polystyrene tubes. Detection limits ranged from 3.10(5) to 3.10(6) molecules, depending on the number of biotins. Multilabelled oligonucleotides have been included into a capture format to detect DNA of Borrelia burgdorferi, the causal agent of Lyme disease. In short, 5'-phosphate oligonucleotides were chemically condensed onto aminated microwells and served to capture PCR-amplified target DNA which, in turn, hybridized with polybiotinylated complementary oligonucleotides. Detection of the hybrids was achieved through streptavidin-conjugated peroxidase and a colorimetric substrate. The solid phase sandwich DNA hybridization assay proved highly specific, very sensitive, rapid and user friendly for the identification of spirochetal DNA in human biological specimens.

Rapid colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA in gastric biopsy specimens.

A very simple, practical, sensitive, and specific colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA is described. This assay, which combines a sensitive sandwich DNA hybridization reaction and a colorimetric protocol similar to those used in conventional enzyme immunoassays, was shown to be suitable for detecting H. pylori-infected gastric biopsy specimens and for monitoring the eradication of the pathogen after treatment. The specificity and sensitivity of the colorimetric hybridization assay were tested by assaying 27 H. pylori strains (4 reference and 23 clinical isolates), 9 strains of other Helicobacter spp. or Campylobacter spp., and 11 clinical isolates of other urease-positive bacteria. The likelihood of H. pylori detection in gastric biopsy specimens by the colorimetric hybridization assay was evaluated with 23 H. pylori-positive and 41 H. pylori-negative biopsy specimens on the basis of positive and negative results, respectively, of culture, rapid urease test, histological examination, and PCR. Biopsy specimens from 33 treated patients, endoscopied 4 to 8 weeks after the end of treatment, were also tested. All H. pylori strains showed positive results in the colorimetric hybridization assay, presenting optical densities at 450 nm (OD450S) of > or = 3.0. None of the other Helicobacter spp., Campylobacter spp., or the clinical isolates of other urease-positive bacteria showed OD450S equal to or greater than the cutoff (mean OD450 cutoff, 0.208). The colorimetric hybridization assay detected all 23 H. pylori-positive biopsy specimens (mean OD450, 2.910 +/- 0.295), while none of the H. pylori-negative biopsy specimens was shown to be positive in the assay (mean OD450, 0.108 +/- 0.025). H. pylori was considered to be not eradicated from three of the posttreatment biopsy specimens by culture, rapid urease test, histological examination, and PCR. They were all positive by the colorimetric hybridization assay, and their OD450S were > or = 3.0. The colorimetric hybridization assay also detected two other H. pylori-positive patients. Specimens from these two patients had negative culture, rapid urease test, and histology results, and a specimen from one of them also tested negative by PCR. These results indicate that the colorimetric hybridization assay is a suitable method both for the diagnosis of H. pylori in biopsy specimens and for the follow-up of patients after the end of treatment.

Cytokeratins are exposed on the outer surface of established human mammary carcinoma cells.

Normal human mammary epithelial cells and established tumour cells of the same origin express three to eight cytokeratins, which are distributed throughout the cytoplasm in the form of intermediate filaments. The combined use of the iodogen and the two-dimensional gel electrophoresis methods has allowed us to demonstrate the presence of cytokeratins 8, 18 and 19 on the outer surface of established human mammary carcinoma cells, in particular MCF-7 cells, while they were absent from the surface of normal mammary cells in primary culture. By ultrastructural immunocytochemistry, these cytokeratins were localized on blebs formed by the cell surface. Cytokeratins 8, 18 and 19 were also detected in the culture medium of mammary carcinoma cells.

[Detection of Borrelia DNA in synovial fluid for diagnosis of Lyme arthritis]. / Nachweis von Borrelien-DNA in der Synovialflüssigkeit zur Diagnose der Lyme-Arthritis.

AIM: To test sensitivity and specificity of a polymerase chain reaction (PCR) targeting the Borrelia specific outer surface protein (Osp) A gene in synovial fluid for the diagnosis of Lyme arthritis, and thus permit an earlier start to treatment. PATIENTS AND METHODS: Prospectively we examined the synovial fluid of 37 patients with the clinical diagnosis of Lyme arthritis or with other arthropathies of known or unknown origin, searching for the presence of detectable borrelial DNA in both arms of the study. Retrospectively we examined the stored synovial fluid from 50 patients of the Department of Rheumatology of the University Hospital, Berne, with the clinical diagnosis of monarthritis or oligoarthritis of unknown etiology, juvenile chronic arthritis or rheumatoid arthritis. The laboratory biologist was unaware of the clinical diagnosis. RESULTS: In the prospective study no true false positive results were found: of the 28 patients without strong clinical suspicion of Lyme arthritis 27 were PCR negative. In one case with positive PCR for borrelial DNA the diagnosis could not be clarified, Lyme arthritis remaining a possibility. Therefore the specificity in the prospective study was at least 96%. Borrelial DNA in the synovial fluid was found in 5 out of 9 patients with strong clinical suspicion of Lyme arthritis. All 7 patients in this group were new, untreated cases. All the 5 PCR positive results belonged to this group, thus the "sensitivity" of the tested method was 71% in untreated cases of Lyme arthritis. In the retrospective study we found borrelial DNA in the synovial fluid of 2 patients. These 2 patients had gonarthritis of unknown origin. Retrospectively these 2 cases could be diagnosed as Lyme arthritis. CONCLUSION: In cases with clinical suspicion of Lyme arthritis the PCR method targeting a borrelial Osp A gene fragment common to all 3 European genospecies shows very good specificity and in untreated cases acceptable sensitivity. Introduction of the method studied into clinical practice is justified.

In vivo deletion of the methicillin resistance mec region from the chromosome of Staphylococcus aureus strains.

Two sets of Staphylococcus aureus isolates recovered from two patients exhibited similar susceptibility profiles except for oxacillin susceptibility (MSSA) or resistance (MRSA). SMA:I macrorestriction and inter-IS256 PCR analysis showed patterns closely related to the Belgian epidemic MRSA clone 1 in each pair of MSSA/MRSA strains. Loss of one large SMA:I DNA fragment and concurrent gain of a smaller fragment in the MSSA isolates was observed. The mecA sequence present in the MRSA was absent in the MSSA variant. Therefore, in vivo deletion of the mec region may occur in some lineages of S. aureus more frequently than previously thought.

Characterization of a C-type retrovirus isolated from an HIV infected cell line: complete nucleotide sequence.

As previously reported, a C-type retrovirus, referred to as retrovirus X was isolated from HIV infected cells. In order to further characterize this virus, the proviral DNA was cloned and sequenced. The organization of the genome (8379 bp) appeared to be typical of the mammalian type C retroviruses. The virus was shown to be closely related to the gibbon ape leukaemia virus (GALV) with 87% similarity when the sequence was compared with the published genome of the Seato strain of GALV. At the level of the long terminal repeat where comparison was possible with other strains, the closest relationship was found with the San Francisco strain of GALV and with the simian sarcoma virus. These results suggest that the isolate should be considered as a strain of GALV.

Characterization of the type III secretion locus of Bordetella pertussis.

Multiple sequence comparisons of proteins of the LcrD/FlbF family allowed the design of primers that specifically amplify sequences coding for type III secretion components. Amplification of Bordetella pertussis DNA with these primers yielded a fragment that was further used as a probe for screening a genomic library. The nucleotide sequence of a positive clone revealed a 2100-bp gene, called bcrD, which specifies a 75-kDa polypeptide homologous to the Yersinia LcrD protein. Chromosome walking allowed the characterization of a 35-kb DNA segment that contains the entire locus and flanking housekeeping genes. The B. pertussis type III secretion locus consists of more than 30 open reading frames (ORFs), most of which are identical to annotated genes of Bordetella spp and share similarities with known type III secretion genes of related bacteria. In order to assess the function of this locus, we engineered a bcrD null mutant. However, none of the tested phenotypes, such as protein secretion, cellular invasion, cytotoxicity or mouse lung colonization, differentiated the mutant from its parental strain. Studies of bcrD and bscN expressions indicated that, under our experimental conditions, these genes are not expressed in vitro. Restriction analyses on pulsed-field gel electrophoresis allowed the type III locus mapping at coordinate position 1,590 kb on the Tohama I strain chromosome.