Label-free DNA detection of hepatitis C virus based on modified conducting polypyrrole films at microelectrodes and atomic force microscopy tip-integrated electrodes

We present a new strategy for the label-free electrochemical detection of DNA hybridization for detecting hepatitis C virus based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes. Synthetic single-stranded 18-mer HCV genotype-1-specific probe DNA has been immobilized at a 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole film established by electropolymerization at the previously formed polypyrrole layer. HCV DNA sequences (244-mer) resulting from the reverse transcriptase-linked polymerase chain reaction amplification of the original viral RNA were monitored by affecting the ion-exchange properties of the polypyrrole film. The performance of this miniaturized DNA sensor system was studied in respect to selectivity, sensitivity, and reproducibility. The limit of detection was determined at 1.82 x 10(-21) mol L-1. Control experiments were performed with cDNA from HCV genotypes 2a/c, 2b, and 3 and did not show any unspecific binding. Additionally, the influence of the spacer length of 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole on the behavior of the DNA sensor was investigated. This biosensing scheme was finally extended to the electrochemical detection of DNA at submicrometer-sized DNA biosensors integrated into bifunctional atomic force scanning electrochemical microscopy probes. The 18-mer DNA target was again monitored by following the ion-exchange properties of the polypyrrole film. Control experiments were performed with 12-base pair mismatched sequences.

Human respiratory syncytial virus detection in children admitted at a community hospital in Botucatu, SP, Brazil

O Vírus Respiratório Sincicial Humano (VRSH) é descrito como o mais importante patógeno viral causador de doenças respiratórias agudas das vias respiratórias inferiores em crianças. Neste estudo 84 amostras de crianças com idade abaixo dos dois anos apresentando sintomas de doença respiratória aguda, foram obtidas no período de setembro de 2000 a novembro de 2001. Analise por imunofluorescência indireta e transcrição reversa seguida de PCR, revelou que 18% (15/84) das amostras foram positivas, sendo que em 80% (12/15) dos casos a detecção de VRSH foi observada em crianças abaixo dos seis meses, e também que os subgrupos A e B co-circularam. Estes são os primeiros dados obtidos para a cidade de Botucatu, sendo que a sazonalidade mostrou-se evidente pela maior circulação desse vírus entre os meses de maio e julho

Detection of cracks in scratching tests in ceramic materials through acoustic emission

The mechanisms of material removal and the interactions among scratches performed in ceramic materials were investigated using acoustic emission signals, and scanning electron microscopy, in scratching experiments. Several testing conditions were used to produce different types of removing mechanism on a glass as well as on a polycrystalline alumina sample composed by heterogeneous grain size. It is known that the material removing process on a polycrystalline ceramic involves intergranular microfracture and grain dislodgement, unlike the chipping produced by the extension of lateral cracks in non-granular materials, such as glass. Distinct settings for velocities, loads, and two types of diamond indenter were tested. The material removal was carried out by three different methods of scratching: single passes, repeated overlapping passes, and parallel scratches. As a general result, there was a clear relationship between the acoustic emission signals and the damage intensity occurred in the material removal. More specifically, there were differences in the acoustic emission signal levels in the scratches made on the alumina and on the glass owing to the material removal mechanisms associated with the structure of these materials. A gradual increase in the acoustic emission levels was observed when the number of repeated passes was increased as a result of the damage accumulation process followed by severe material removal. It was also noticed that the acoustic emission signals were capable of reflecting the interactions between two parallel scratches.

Label-free DNA detection based on modified conducting polypyrrole films at microelectrodes

A label-free electrochemical detection method for DNA hybridization based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes is reported. Synthetic single-stranded 27-mer oligonucleotides (probe) have been immobilized at 2,5-bis(2-thienyl)-N-(3-phosphorylpropyl)pyrrole film formed by electropolymerization on the previously formed polypyrrole layer. The 27- or 18-mer target oligonucleotides were monitored via the electrochemically driven anion exchange of the inner polypyrrole film. The performance of the miniaturized DNA biosensor system was studied in respect to selectivity, sensitivity, reproducibility, and regeneration of the sensor. Control experiments were performed with a noncomplementary target of 27-mer DNA and 12 base-pair mismatched 18-mer sequences, respectively, and did not show any unspecific binding. Under optimized experimental conditions, the label-free electrochemical biosensor enabled the detection limits of 0.16 and 3.5 fmol for the 18- and 2 7-mer DNA strand, respectively. Furthermore, we demonstrate reusability of the electrochemical DNA biosensor after successful recovery of up to 100% of the original signal by regenerating the DNA label-free electrode with 50 mM HCl at room temperature.

Evaluation of a larval development assay for the detection of anthelmintic resistance in Ostertagia circumcincta

Two experiments were carried out to evaluate a larval development assay for the detection of anthelmintic resistance in O. circumcincta. In Experiment I, the dose responses to levamisole (LEV), thiabendazole (TBZ) and ivermectin (IVM) of 8 isolates of O. circumcincta were measured 34 days after infection (DAI). Four of these isolates were shown to be resistant to 1 or more anthelmintics. With 2 exceptions, all isolates considered to be resistant had higher LD50 values than the susceptible isolates for that anthelmintic. One exception was isolate RM8, which was considered to be resistant to all 3 anthelmintics based on faecal egg count reduction tests in goats, but the LD50 value for LEV did not differ from that for the susceptible isolates. The other exception was an isolate considered to be susceptible to TBZ which had a relatively high LD50 value. In an unrelated trial that was prompted by this finding, this isolate was confirmed to be benzimidazole-resistant. Isolate RM8 and an isolate susceptible to all 3 anthelmintics (SK2) were used in the second experiment, which was conducted to monitor changes in the LD50 values of LEV, TBZ and IVM over time following a single infection of 35 000 infective larvae in young sheep. Faecal samples were collected weekly from 24 to 115 DAI. With all 3 anthelmintics, the LD50 values increased with time to a peak around 50-60 DAI, and then declined to levels similar to those observed soon after patency. This trend was consistent for both isolates. The highest mean LD50 values for isolates SK2 for IVM and TBZ and RM8 for IVM and RM8, respectively, were 1.7 and 1.8 times, and 2.2 and 2.9 times higher than the initial mean LD50 values. There was a clear distinction in LD50 values between isolates at each sampling day for both IVM and TBZ. However, as a consequence of the changes in LD50 values with time, the peak LD50 values of IVM for isolate SK2 were higher than the minimum LD50 values of isolate RM8. As there was no apparent difference in LEV efficacy between these 2 isolates, the data were pooled. The highest mean LD50 value was 2.3 times higher than the initial LD50 value. (C) 1997 Australian Society for Parasitology.

Epstein-Barr Virus (EBV) detection and typing by PCR: a contribution to diagnostic screening of EBV-positive Burkitt's lymphoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular and Serologic Detection of Ehrlichia spp. in Endangered Brazilian Wild Captive Felids

Ehrlichiosis, an emergent tickborne disease that affects both humans and animals, may represent a threat to the survival and preservation of wild felids in Brazil There are few studies of ehrlichiosis in wild felids in Brazil, but Ehrlichia spp are present in domestic cats Antibodies to Ehrlichia canis have been reported in a puma (Puma concolor) In this study we assessed the presence of these hemoparasites in the blood of Brazilian wild captive felids of the 72 animals tested, 5 (7%) were seropositive for the E cams antigen, and L1 (15%) were positive for E emirs DNA sequences We also performed sequence alignment to establish the identity of the parasite species infecting these animals using 16S rRNA and omp-1 genes Sequences based on 16S rRNA were similar to those found in dogs and cats from Thailand, Brazil, China, and Taiwan and with E canis obtained from a single individual (human) in Venezuela Ehrlichia sp sequence from sampled felines based on omp-1. gene was similar to the p28 and p30 multigene family of E canis To our knowledge, this is the first study of molecular detection of Ehrlichia sp in Brazilian wild feline species

A single multiplex PCR and SNaPshot minisequencing reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P > 0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P < 0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P > 0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P < 0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P < 0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar. © 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Tethered DNA scaffolds on optical sensor platforms for detection of hipO gene from Campylobacter jejuni

DNA biosensors have gained increased attention over traditional diagnostic methods due to their fast and responsive operation and cost-effective design. The specificity of DNA biosensors relies on single-stranded oligonucleotide probes immobilized to a transduction platform. Here, we report the development of biosensors to detect the hippuricase gene (hipO) from Campylobacter jejuni using direct covalent coupling of thiol- and biotin-labeled single-stranded DNA (ssDNA) on both surface plasmon resonance (SPR) and diffraction optics technology (DOT, dotLab) transduction platforms. This is the first known report of the dotLab to detect targeted DNA. Application of 6-mercapto-1-hexanol as a spacer thiol for SPR gold surface created a self-assembled monolayer that removed unbound ssDNA and minimized non-specific detection. The detection limit of SPR sensors was shown to be 2.5 nM DNA while dotLab sensors demonstrated a slightly decreased detection limit of 5.0 nM (0.005 μM). It was possible to reuse the SPR sensor due to the negligible changes in sensor sensitivity (∼9.7 × 10 -7 ΔRU) and minimal damage to immobilized probes following use, whereas dotLab sensors could not be reused. Results indicated feasibility of optical biosensors for rapid and sensitive detection of the hipO gene of Campylobacter jejuni using specific ssDNA as a probe. © 2011 Elsevier B.V.

A single multiplex SNaPshot reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. DNA was extracted from blood spotted on filter paper using Chelex protocol. Forty SNPs were selected targeting haplogroup-specific mutations in Europeans, Africans and Asians (only precursors of Native Americans haplogroups A2, B2, C1, and D1) and two non-coding SNPs were chosen to increase the power of discrimination between individuals (SNPs positions 16,519 and 16,362). It was done using a modified version of a previously published multiplex SNaPshot minisequencing reaction established to resolve European haplogroups, adding SNPs targeting Africans (L0, L1, L2, L3, and L*) and Asians (A, B, C, and D) haplogroups based on SNPs described at PhyloTree.org build 2. PCR primers were designed using PerlPrimer software and checked with the Autodimer program. Thirty-three primer-pairs were used to amplify 42 SNPs. Using this panel, we were able to successfully classify 160 individuals into their correct haplogroups. Complete SNP profiles were obtained from 10. pg of total DNA. We conclude that it is possible to build and genotype more than 40 mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations. © 2011.

Point vibration measurements for the detection of shallow-buried objects

A major UK initiative, entitled 'Mapping the Underworld', is seeking to address the serious social, environmental and economic consequences arising from an inability to locate accurately and comprehensively the buried utility service infrastructure without resorting to extensive excavations. Mapping the Underworld aims to develop and prove the efficacy of a multi-sensor device for accurate remote buried utility service detection, location and, where possible, identification. One of the technologies to be incorporated in the device is low-frequency vibro-acoustics, and application of this technique for detecting buried infrastructure is currently being investigated. Here, the potential for making a number of simple point vibration measurements in order to detect shallow-buried objects, in particular plastic pipes, is explored. Point measurements can be made relatively quickly without the need for arrays of surface sensors, which can be expensive, time-consuming to deploy, and sometimes impractical in congested areas. At low frequencies, the ground behaves as a simple single-degree-of-freedom (mass-spring) system with a well-defined resonance, the frequency of which will depend on the density and elastic properties of the soil locally. This resonance will be altered by the presence of a buried object whose properties differ from the surrounding soil. It is this behavior which can be exploited in order to detect the presence of a buried object, provided it is buried at a sufficiently shallow depth. The theoretical background is described and preliminary measurements are made both on a dedicated buried pipe rig and on the ground over a domestic waste pipe. Preliminary findings suggest that, for shallow-buried pipes, a measurement of this kind could be a quick and useful adjunct to more conventional methods of buried pipe detection. © 2012 Elsevier Ltd. All rights reserved.

Detection of buried pipes using a shear wave ground surface vibration technique

A major UK initiative, entitled 'Mapping the Underworld', is seeking to address the serious social, environmental and economic consequences arising from an inability to locate the buried utility service infrastructure without resorting to extensive excavations. Mapping the Underworld aims to develop and prove the efficacy of a multi-sensor device for accurate remote buried utility service detection, location and, where possible, identification. One of the technologies to be incorporated in the device is low-frequency vibro-acoustics, and the application of this technology for detecting buried infrastructure is currently being investigated. Here, a shear wave ground vibration technique for detecting buried pipes is described. For this technique, shear waves are generated at the ground surface, and the resulting ground surface vibrations measured, using geophones, along a line traversing the anticipated run of the pipe. Measurements were made at a test site with a single pressurized polyethylene mains water pipe. Time-extended signals were employed to generate the illuminating wave. Cross-correlation functions between the measured ground velocities and a reference measurement adjacent to the excitation were then calculated and summed using a stacking method to generate a cross-sectional image of the ground. The wide cross-correlation peaks caused by high ground attenuation were partially compensated for by using a generalized cross-correlation function called the smoothed coherence transform. To mitigate the effects of other potential sources of vibration in the vicinity, the excitation signal was used as an additional reference when calculating the generalized cross-correlation functions. For two out of three tests, the pipe was detected, indicating that this technique will be a valuable addition to the Mapping the Underworld armoury.

The detection of Cryptosporidium serpentis in snake fecal samples by real-time PCR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Semi-supervised learning with concept drift using particle dynamics applied to network intrusion detection data

Concept drift, which refers to non stationary learning problems over time, has increasing importance in machine learning and data mining. Many concept drift applications require fast response, which means an algorithm must always be (re)trained with the latest available data. But the process of data labeling is usually expensive and/or time consuming when compared to acquisition of unlabeled data, thus usually only a small fraction of the incoming data may be effectively labeled. Semi-supervised learning methods may help in this scenario, as they use both labeled and unlabeled data in the training process. However, most of them are based on assumptions that the data is static. Therefore, semi-supervised learning with concept drifts is still an open challenging task in machine learning. Recently, a particle competition and cooperation approach has been developed to realize graph-based semi-supervised learning from static data. We have extend that approach to handle data streams and concept drift. The result is a passive algorithm which uses a single classifier approach, naturally adapted to concept changes without any explicit drift detection mechanism. It has built-in mechanisms that provide a natural way of learning from new data, gradually "forgetting" older knowledge as older data items are no longer useful for the classification of newer data items. The proposed algorithm is applied to the KDD Cup 1999 Data of network intrusion, showing its effectiveness.