Performance of RT-PCR-ELISA for the detection of peste des petits ruminants virus

A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR-ELISA) was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the amplification/probing of the 'N' gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer binding sites of the 'N' gene sequences available and the ability to amplify PPR viral genome from different sources of samples. RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR followed by agarose gel based detection of PCR product. Based on the Mean (mean +/- 3S.D.) optical density (OD) values of 47 RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54% positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6 days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node, lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of RT-PCR products are discussed. (c) 2006 Elsevier B.V. All rights reserved.

A Wavelet based Teager Energy Operator for Spike Detection in Microelectrode Array Recordings

Spike detection in neural recordings is the initial step in the creation of brain machine interfaces. The Teager energy operator (TEO) treats a spike as an increase in the `local' energy and detects this increase. The performance of TEO in detecting action potential spikes suffers due to its sensitivity to the frequency of spikes in the presence of noise which is present in microelectrode array (MEA) recordings. The multiresolution TEO (mTEO) method overcomes this shortcoming of the TEO by tuning the parameter k to an optimal value m so as to match to frequency of the spike. In this paper, we present an algorithm for the mTEO using the multiresolution structure of wavelets along with inbuilt lowpass filtering of the subband signals. The algorithm is efficient and can be implemented for real-time processing of neural signals for spike detection. The performance of the algorithm is tested on a simulated neural signal with 10 spike templates obtained from [14]. The background noise is modeled as a colored Gaussian random process. Using the noise standard deviation and autocorrelation functions obtained from recorded data, background noise was simulated by an autoregressive (AR(5)) filter. The simulations show a spike detection accuracy of 90%and above with less than 5% false positives at an SNR of 2.35 dB as compared to 80% accuracy and 10% false positives reported [6] on simulated neural signals.

sRNAscanner: A Computational Tool for Intergenic Small RNA Detection in Bacterial Genomes

Background:Bacterial non-coding small RNAs (sRNAs) have attracted considerable attention due to their ubiquitous nature and contribution to numerous cellular processes including survival, adaptation and pathogenesis. Existing computational approaches for identifying bacterial sRNAs demonstrate varying levels of success and there remains considerable room for improvement. Methodology/Principal Findings: Here we have proposed a transcriptional signal-based computational method to identify intergenic sRNA transcriptional units (TUs) in completely sequenced bacterial genomes. Our sRNAscanner tool uses position weight matrices derived from experimentally defined E. coli K-12 MG1655 sRNA promoter and rho-independent terminator signals to identify intergenic sRNA TUs through sliding window based genome scans. Analysis of genomes representative of twelve species suggested that sRNAscanner demonstrated equivalent sensitivity to sRNAPredict2, the best performing bioinformatics tool available presently. However, each algorithm yielded substantial numbers of known and uncharacterized hits that were unique to one or the other tool only. sRNAscanner identified 118 novel putative intergenic sRNA genes in Salmonella enterica Typhimurium LT2, none of which were flagged by sRNAPredict2. Candidate sRNA locations were compared with available deep sequencing libraries derived from Hfq-co-immunoprecipitated RNA purified from a second Typhimurium strain (Sittka et al. (2008) PLoS Genetics 4: e1000163). Sixteen potential novel sRNAs computationally predicted and detected in deep sequencing libraries were selected for experimental validation by Northern analysis using total RNA isolated from bacteria grown under eleven different growth conditions. RNA bands of expected sizes were detected in Northern blots for six of the examined candidates. Furthermore, the 5'-ends of these six Northern-supported sRNA candidates were successfully mapped using 5'-RACE analysis. Conclusions/Significance: We have developed, computationally examined and experimentally validated the sRNAscanner algorithm. Data derived from this study has successfully identified six novel S. Typhimurium sRNA genes. In addition, the computational specificity analysis we have undertaken suggests that similar to 40% of sRNAscanner hits with high cumulative sum of scores represent genuine, undiscovered sRNA genes. Collectively, these data strongly support the utility of sRNAscanner and offer a glimpse of its potential to reveal large numbers of sRNA genes that have to date defied identification. sRNAscanner is available from: http://bicmku.in:8081/sRNAscanner or http://cluster.physics.iisc.ernet.in/sRNAscanner/.

Development of a probe-based blotting technique for the detection of Tobacco streak virus

Digoxigenin (DIG)-labeled DNA probe was developed for a sensitive and rapid detection of the Tobacco streak virus (TSV) isolates in India by dot-blot and tissue print hybridization techniques. DIG-labeled DNA probe complementary to the coat protein (CP) region of TSV sunflower isolate was designed and used to detect the TSV presence at field levels. Dot-blot hybridization was used to check a large number of TSV isolates with a single probe. In addition, a sensitivity of the technique was examined with the different sample extraction methods. Another technique, the tissue blot hybridization offered a simple, reliable procedure and did not require a sample processing. Thus, both non-radioactively labeled probe techniques could facilitate the sample screening during TSV outbreaks and offer an advantage in quarantine services.

Detection and characterisation of circulating cysticercal antigens in human cerebrospinal fluid

With biotin labelled and unlabelled immunoglobulin fraction of anticysticercal antibodies raised in rabbits, tandem-enzyme linked immunosorbent assay (T-ELISA), capture-dot immunobinding assay (C-DIA) and reverse passive haemagglutination (RPHA) tests were developed for the detection of cysticercal antigens. The sensitivity levels were respectively, 9 ng ml−1, 2 ng ml−1 and 45 ng ml−1. All three methods were of equal specificity as none of the antigens of Mycobacterium tuberculosis, Japanese encephalitis virus and Echinococcus granulosus reacted with anticysticercal IgG. Cysticercal antigens were detected in the cerebrospinal fluid (CSF) of confirmed neurocysticercosis at sensitivity levels of 91·6% by T-ELISA, 83·33% by C-DIA and 75% by RPHA and specificity levels of >93%. Western analysis of these antigens in CSF showed mainly antigens of 64–68 kDa and 24–28 kDA. By crossed immunoelectrophoresis (CIE) with an intermediate gel technique, five circulating antigens were found to be released from scolex and fluid.

Enhanced sensitivity for the differential pulse polarographic determination of molybdenum(VI) based on adsorption catalytic current

A differential pulse polarographic (DPP) method based on the adsorption catalytic current in a medium containing chlorate and 8-hydroxyquinoline (oxine) is suggested for the determination of molybdenum(VI). Experimental conditions such as pH and the composition of supporting electrolyte have been optimized to get a linear calibration graph at trace levels of Mo(VI). The sensitivity for molybdenum can be considerably enhanced by this method. The influence of possible interferences on the catalytic current has been investigated. The sensitivity of the method is compared with those obtained for other DPP methods for molybdenum. A detection limit of 1.0 x 10(-8) mol/L has been found.

Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India

Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV AP The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Carbon based materials for low temperature detection

Resistance temperature detectors (RTDs) are being widely used to detect low temperature, while thermocouples (TCs) are being used to detect high temperature. The materials suitable for RTDs are platinum, germanium, carbon, carbon-glass, cernox, etc. Here, we have reported the possible application of another form of carbon i.e. carbon nanotubes in low temperature thermometry. It has been shown the resistance R and the sensitivity of carbon nanotube bundles can be tuned and made suitable for ultralow temperature detection. We report on the R-T measurement of carbon nanotube bundles from room temperature down to 1 K to felicitate the possible application of bundles in low temperature RTDs. ©2008 American Institute of Physics

Antigen detection assay with parasite specific monoclonal antibodies for diagnosis of lymphatic filariasis

Background: Lymphatic filariasis is a painful and profoundly disfiguring disease. Infection is usually acquired in childhood but its visible manifestations occur later in life, causing temporary or permanent disability. The importance of developing effective assays to diagnose, monitor and evaluate human lymphatic filariasis has been emphasized by the WHO. Methods: High-affinity monoclonal antibodies (mAbs) specific for recombinant filarial antigen WbSXP-1 were developed. An ELISA based capture assay using monoclonal and polyclonal antibodies for WbSXP-1 was used for detection of circulating filarial antigen. Results: High-affinity monoclonal antibodies (mAbs) were developed that specifically binds both W. bancrofti and B. malayi mf antigens. Two mAbs (1F6H3 and 2E12E3) of subclass IgG2a and IgM showed high affinity, avidity and reactivity to recombinant and mf native antigen. Both the mAbs were used in combination as capture antibodies and polyclonal as detection antibody to develop the assay. The assay showed very high sensitivity towards W. bancrofti mf positive samples compared to endemic normal samples (P<0.0001). Conclusion: A capture assay using high-affinity monoclonal antibodies for WbSXP-1 was developed for the detection of filarial circulating antigen in clinical samples from bancroftian infection. Besides, this would also help in epidemiological studies in endemic areas of filarial infections. (C) 2011 Elsevier B.V. All rights reserved.

Direct detection of Salmonella without pre-enrichment in milk, ice-cream and fruit juice by PCR against hilA gene

A novel PCR based assay was devised to specifically detect contamination of any Salmonella serovar in milk, fruit juice and ice-cream without pre-enrichment. This method utilizes primers against hilA gene which is conserved in all Salmonella serovars and absent from the close relatives of Salmonella. An optimized protocol, in terms time and money, is provided for the reduction of PCR contaminants from milk, ice-cream and juice through the use of routine laboratory chemicals. The simplicity, efficiency (time taken 3-4 h) and sensitivity (to about 5-10 CFU/ml) of this technique confers a unique advantage over other previously used time consuming detection techniques. This technique does not involve pre-enrichment of the samples or extensive sample processing, which was a pre-requisite in most of the other reported studies. Hence, this assay can be ideal for adoption, after further fine tuning, by food quality control for timely detection of Salmonella contamination as well as other food-borne pathogens (with species specific primers) in food especially milk, ice-cream and fruit juice. (C) 2011 Elsevier Ltd. All rights reserved.

Detection and determination of manganese concentration in water using a fiber Bragg grating coupled with nanotechnology

Through this paper we experimentally demonstrate the fabrication of a fiber Bragg grating (FBG) chemical sensor to detect and determine the manganese concentration in water and compare our results with sophisticated spectroscopic methods, such as atomic absorption spectrometry and the inductively coupled plasma method. Here we propose a simple method to develop a thin layer of gold nanoparticles above the etched grating region to enhance the sensitivity of the reflected spectrum of the FBG. By doing so, we achieve a sensitivity of 1.26 nm/parts per million in determining the trace level of Mn in water. Proper reagents are used to detect manganese in water. (C) 2011 Optical Society of America

Femtogram Detection of Explosive Nitroaromatics: Fluoranthene-Based Fluorescent Chemosensors

Herein we report a novel fluoranthene-based fluorescent fluorophore 7,10-bis(4-bromophenyl)-8,9-bis4-(hexyloxy)phenyl]fluoranthene (S-3) and its remarkable properties in applications of explosive detection. The sensitivity towards the detection of nitroaromatics (NACs) was evaluated through fluorescence quenching in solution, vapor, and contact mode approaches. The contact mode approach using thin-layer silica chromatograp- hic plates exhibited a femtogram (1.15 fg cm(-2)) detection limit for trinitrotoluene (TNT) and picric acid (PA), whereas the solution-phase quenching showed PA detection at the 2-20 ppb level. Fluorescence lifetime measurements revealed that the quenching is static in nature and the quenching process is fully reversible. Binding energies between model binding sites of the S-3 and analyte compounds reveal that analyte molecules enter into the cavity created by substituted phenyl rings of fluoranthene and are stabilized by strong intermolecular interactions with alkyl chains. It is anticipated that the sensor S-3 could be a promising material for the construction of portable optical devices for the detection of onsite explosive nitroaromatics.

Employing denaturation for rapid electrochemical detection of myoglobin using TiO2 nanotubes

An alternative antibody-free strategy for the rapid electrochemical detection of cardiac myoglobin has been demonstrated here using hydrothermally synthesized TiO2 nanotubes (Ti-NT). The denaturant induced unfolding of myoglobin led to easy access of the deeply buried electroactive heme center and thus the efficient reversible electron transfer from protein to electrode surface. The sensing performance of the Ti-NT modified electrodes were compared vis a vis commercially available titania and GCEs. The tubular morphology of the Ti-NT led to facile transfer of electrons to the electrode surface, which eventually provided a linear current response (obtained from cyclic voltammetry) over a wide range of Mb concentration. The sensitivity of the Ti-NT based sensor was remarkable and was equal to 18 mu A mg(-1) ml (detection limit = 50 nM). This coupled with the rapid analysis time of a few tens of minutes (compared to a few days for ELISA) demonstrates its potential usefulness for the early detection of acute myocardial infarction (AMI).

A Guided Wave approach for the detection of damage in a structure having elements with periodic damage

A wave propagation based approach for the detection of damage in components of structures having periodic damage has been proposed. Periodic damage pattern may arise in a structure due to periodicity in geometry and in loading. The method exploits the Block-Floquet band formation mechanism, a feature specific to structures with periodicity, to identify propagation bands (pass bands) and attenuation bands (stop bands) at different frequency ranges. The presence of damage modifies the wave propagation behaviour forming these bands. With proper positioning of sensors a damage force indicator (DFI) method can be used to locate the defect at an accuracy level of sensor to sensor distance. A wide range of transducer frequency may be used to obtain further information about the shape and size of the damage. The methodology is demonstrated using a few 1-D structures with different kinds of periodicity and damage. For this purpose, dynamic stiffness matrix is formed for the periodic elements to obtain the dispersion relationship using frequency domain spectral element and spectral super element method. The sensitivity of the damage force indicator for different types of periodic damages is also analysed.

D-A-D-structured conducting polymer-modified electrodes for detection of lead(II) ions in water

Donor-acceptor-donor-structured thiophene derivative-based conducting polymer poly(7,9-dithiophene-2yl-8H-cyclopentaa]acenaphthalene-8-one) was chemically synthesized. This polymer was used to modify both glassy-carbon and carbon-paste electrode, which was used to detect lead(II) ions present in water in the range of 1 mM to 0.1 mu M. Cyclic voltammetry confirms the formation of the co-ordination complex between the soft segment of polymer and the dissolved lead ion. Anodic stripping voltammetry was carried out by the modified electrode to determine the lower limit of detection of dissolved lead(II) species in the solution. Differential adsorptive stripping and impedance measurements were also conducted to find the lowest possible response of the as-synthesized polymer to lead(II) ion in water. The electrochemical performance of the modified electrodes at different pH (4, 7 and 9) environments was carried out by stripping voltammetry, to get optimum sensitivity and stability under these conditions. Finally, interference analysis was carried out to detect the modified electrode's sensitivity towards lead ion affinity in water.