Increased Detection Sensitivity Research Articles

Innovative tools for detection of plant pathogenic viruses and bacteria

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.

Increased Sensitivity in PCR Detection of tdh-Positive Vibrio parahaemolyticus in Seafood with Purified Template DNA

PCR is an important method for the detection of thermostable direct hemolysin gene (tdh)–positive (pathogenichemolysin-producing) strains of Vibrio parahaemolyticus in seafood because tdh-negative (nonpathogenic) V. parahaemolyticus strains often contaminate seafood and interfere with the direct isolation of tdh-positive V. parahaemolyticus. In this study, the use of PCR to detect the tdh gene of V. parahaemolyticus in various seafoods artificially contaminated with tdh-positive V. parahaemolyticus was examined. PCR was inhibited by substances in oysters, squid, mackerel, and yellowtail but not by cod, sea bream, scallop, short-necked clam, and shrimp. To improve detection, DNA was purified by either the silica membrane method, the glass fiber method, or the magnetic separation method, and the purified DNA was used as the PCR primer template. For all samples, the use of the silica membrane method and the glass fiber method increased detection sensitivity. The results of this study demonstrate that the use of properly purified template DNA for PCR markedly increases the effectiveness of the method in detecting pathogenic tdh-positive V. parahaemolyticus in contaminated seafood.

Separation and on-line concentration of bisphenol A and alkylphenols by micellar electrokinetic chromatography with anionic surfactant

Separation and on-line concentration of bisphenol A and three alkylphenols were investigated by micellar electrokinetic chromatography with the anionic surfactant, sodium dodecyl sulfate. The separation conditions were optimized by the simultaneous addition of the organic solvent and cyclodextrin to the running solution. The separation of hydrophobic analytes and 4-nonylphenol isomers was improved by the addition of 10% methanol and 5 m M β-cyclodextrin to the running solution. When the sweeping with the running solution was used as the on-line concentration procedure, 69-, 48-, 55- and 41-fold increases in detection sensitivity were obtained for bisphenol A, 4- tert.-butylphenol and 4-(1,1,3,3-tetramethylbutyl)phenol, and the second peak of 4-nonylphenol isomers, respectively. The detection limits were 0.0071, 0.0065, 0.021 and 0.055 mg/l, respectively. These results were better than those with the cationic surfactant, tetradecyltrimethylammonium bromide.

A novel microelectronic gas sensor utilizing carbon nanotubes for hydrogen gas detection

A novel microelectronic gas sensor utilizing carbon nanotubes (CNTs) in a thin-layered Pd/ CNTs/ n +- Si structure for hydrogen detection has been achieved. The sensor is fabricated on an n-type silicon wafer, which is needed as an ohmic supporting substrate. Multiwalled CNTs were grown selectively on the substrate via catalytic activation with microwave plasma enhanced chemical vapor deposition. The I– V characteristics of the sensor exhibit Schottky diode behavior at room temperature with marked sensitivity or current changes in the presence of hydrogen. Increasing detection sensitivity in hydrogen sensing was observed with increasing operating temperature. The results demonstrate that CNTs configured as a gas sensor has high sensitivity to hydrogen over a wide temperature range. Behaviors of the sensor in the presence of hydrogen and at elevated temperature were discussed. The successful utilization of CNTs in gas sensors may open a new door for the development of novel nanostructure gas-sensing devices.

Tandem isotachophoresis-zone electrophoresis via base-mediated destacking for increased detection sensitivity in microfluidic systems.

Electrophoresis in microfluidic devices is becoming a useful analytical platform for a variety of biological assays. In this report, we present a method that allows for an increased sensitivity of detection of fluorescent molecules in microfluidic electrophoresis devices. This capability is provided by the implementation of a particular buffer system that is designed to initially function in an isotachophoretic (ITP) mode and, then after a controlled amount of electric current has been applied to the system, to transition to a zone electrophoretic mode. In the initial ITP mode, analytes dissolved in a large volume of injected sample are concentrated into a single narrow zone. After application of a sufficient and adjustable amount of electric current, the system switches into a zone electrophoretic mode, where the concentrated analytes are separated according to their electrophoretic mobilities. Application of this tandem ITP-zone electrophoretic strategy to the concentration, separation, and detection of fluorescent reporter molecules in a standard microfluidic device results in an approximately 50-fold increase in detection sensitivity relative to equivalent separations that are obtained with zone electrophoresis alone. Even with very long initial sample plugs (up to 3000 microm), this strategy produces electrophoretic separations with high resolutions and peak efficiencies. This strategy can be implemented to increase detection sensitivity in any standard microfluidic electrophoresis platform and does not require any specialized hardware or microchannel configurations.

DNA probe attachment on plastic surfaces and microfluidic hybridization array channel devices with sample oscillation

DNA probe immobilization on plastic surfaces and device assembly are both critical to the fabrication of microfluidic hybridization array channel (MHAC) devices. Three oligonucleotide (oligo) probe immobilization procedures were investigated for attaching oligo probes on four different types of plastic surfaces (polystyrene, polycarbonate, poly(methylmethacrylate), and polypropylene). These procedures are the Surmodics procedure, the cetyltrimethylammonium bromide (CTAB) procedure, and the Reacti-Bind procedure. To determine the optimal plastic substrate and attachment chemistry for array fabrication, we investigated plastic hydrophobicity, intrinsic fluorescence, and oligo attachment efficiency. The Reacti-Bind procedure is least effective for attaching oligo probes in the microarray format. The CTAB procedure performs well enough to use in array fabrication, and the concentration of CTAB has a significant effect on oligo immobilization efficiency. We also found that use of amine-modified oligo probes resulted in better immobilization efficiency than use of unmodified oligos with the CTAB procedure. The oligo probe immobilization on plastic surfaces by the Surmodics procedure is the most effective with regard to probe spot quality and hybridization sensitivity. A DNA hybridization assay on such a device results in a limit of detection of 12 pM. Utilizing a CO 2 IR laser machining and adhesive layer approach, we have developed an improved procedure for realizing a DNA microarray inside a microfluidic channel. This device fabrication procedure allows for more feasible spot placement in the channel and reduced sample adsorption by adhesive tapes used in the fabrication procedure. We also demonstrated improved hybridization kinetics and increased detection sensitivity in MHAC devices by implementing sample oscillation inside the channel. A limit of detection of 5 pM has been achieved in MHAC devices with sample oscillation.

Using temporal context to improve biosurveillance

Current efforts to detect covert bioterrorist attacks from increases in hospital visit rates are plagued by the unpredictable nature of these rates. Although many current systems evaluate hospital visit data 1 day at a time, we investigate evaluating multiple days at once to lessen the effects of this unpredictability and to improve both the timeliness and sensitivity of detection. To test this approach, we introduce simulated disease outbreaks of varying shapes, magnitudes, and durations into 10 years of historical daily visit data from a major tertiary-care metropolitan teaching hospital. We then investigate the effectiveness of using multiday temporal filters for detecting these simulated outbreaks within the noisy environment of the historical visit data. Our results show that compared with the standard 1-day approach, the multiday detection approach significantly increases detection sensitivity and decreases latency while maintaining a high specificity. We conclude that current biosurveillance systems should incorporate a wider temporal context to improve their effectiveness. Furthermore, for increased robustness and performance, hybrid systems should be developed to capitalize on the complementary strengths of different types of temporal filters.

Early Warning Fire Detection System Using a Probabilistic Neural Network

The Navy program, Damage Control-Automation for Reduced Manning is focused on enhancing automation of ship functions and damage control systems. A key element to this objective is the improvement of current fire detection systems. As in many applications, it is desired to increase detection sensitivity and,more importantly increase the reliability of the detection system through improved nuisance alarm immunity. Improved reliability is needed, such that fire detection systems can automatically control fire suppression systems. The use of multi-criteria based detection technology continues to offer the most promising means to achieve both improved sensitivity to real fires,and reduced susceptibility to nuisance alarm sources. A multi-criteria early warning fire detection system, has been developed to provide reliable warning of actual fire conditions, in less time, with fewer nuisance alarms,than can be achieved with commercially available smoke detection systems. In this study a four-sensor array and a Probabilistic Neural Network have been used to produce an early warning fire detection system. A prototype early warning fire detector was built and tested in a shipboard environment. The current alarm algorithm resulted in better overall performance than the commercial smoke detectors, by providing both improved nuisance source immunity with generally equivalent or faster response times.

Separation and on-line concentration of bisphenol A and alkylphenols by micellar electrokinetic chromatography with cationic surfactant.

The separation and on-line concentration of bisphenol A and three alkylphenols were investigated by micellar electrokinetic chromatography with cationic surfactant. Tetradecyltrimethylammonium bromide was used as surfactant and the separation conditions were optimized by the addition of the organic solvents and cyclodextrins to the running solution. The separation of hydrophobic analytes and 4-nonylphenol isomers was improved by the addition of 20% acetonitrile and 20 mM beta-cyclodextrin to the running solution. When the sweeping with the running solution used as the on-line concentration procedure, 56-, 67- and 29-fold increase in detection sensitivity of bisphenol A, 4-tert.-butylphenol and 4-(1,1,3,3-tetramethylbutyl)phenol, respectively. The detection limits were 0.030, 0.098 and 0.159 mg/l, respectively.

Sensitivity of multiplex real-time PCR reactions, using the LightCycler and the ABI PRISM 7700 Sequence Detection System, is dependent on the concentration of the DNA polymerase

The introduction of multiplex PCR techniques to clinical laboratories has provided a means to streamline assays and to produce multiple results with minimal effort. While this methodology is very beneficial, care must be taken to ensure that reactions are properly optimized to allow for maximum sensitivity. This study was conducted to determine whether the sensitivity of multiplex-real-time PCR assays could be improved by increasing the concentration of DNA polymerase within a reaction. Multiplex reactions were designed to simultaneously detect the human HLA-DQ gene and a sequence from the UL83 region of the CMV genome. Two real-time PCR systems, one utilizing AmpliTaq Gold DNA polymerase and the ABI 7700 Sequence Detection System, and one utilizing FastStart Taq DNA polymerase and the Roche LightCycler were tested. The results indicated that increasing the AmpliTaq Gold concentration from 0·050 to 0·10U/μl and the FastStart Taq concentration from 0·1875 to 0·375U/μl increased detection sensitivity from 5000 to 50CMV copies per PCR reaction. In separate experiments, commercially prepared mastermixes were utilized for both real-time PCR platforms as per the manufacturer's suggestions or with the addition of supplemental DNA polymerase. In assays designed to detect 4CMV genome copies per reaction, the addition of 2·5U of AmpliTaq Gold to TaqMan Universal Mastermix increased the detection rate from 21 to 67%, and the addition of 5U of FastStart Taq to FastStart DNA Master Hybridization Probes mastermix increased the detection rate from 17 to 56%. These results indicate that increasing the DNA polymerase concentration in multiplex real-time PCR reactions may be a simple way to optimize assay sensitivity.

Particle detection, number estimation, and feature measurement in gene transfer studies: optical fractionator stereology integrated with digital image processing and analysis

Assessing the efficacy of in vivo gene transfer often requires a quantitative determination of the number, size, shape, or histological visualization characteristics of biological objects. The optical fractionator has become a choice stereological method for estimating the number of objects, such as neurons, in a structure, such as a brain subregion. Digital image processing and analytic methods can increase detection sensitivity and quantify structural and/or spectral features located in histological specimens. We describe a hardware and software system that we have developed for conducting the optical fractionator process. A microscope equipped with a video camera and motorized stage and focus controls is interfaced with a desktop computer. The computer contains a combination live video/computer graphics adapter with a video frame grabber and controls the stage, focus, and video via a commercial imaging software package. Specialized macro programs have been constructed with this software to execute command sequences requisite to the optical fractionator method: defining regions of interest, positioning specimens in a systematic uniform random manner, and stepping through known volumes of tissue for interactive object identification (optical dissectors). The system affords the flexibility to work with count regions that exceed the microscope image field size at low magnifications and to adjust the parameters of the fractionator sampling to best match the demands of particular specimens and object types. Digital image processing can be used to facilitate object detection and identification, and objects that meet criteria for counting can be analyzed for a variety of morphometric and optical properties.

Orthogonal Polynomial Regression for the Detection of Response Variability in Event-Related fMRI

Previous studies using event-related potentials (ERPs) have identified a variety of brain regions that respond to rare nontarget distractor stimuli presented in the visual oddball task. By contrast, event-related functional magnetic resonance imaging (ER-fMRI) studies using similar stimuli have found little or no response in these same regions, suggesting that ER-fMRI may be less sensitive than ERPs for detecting stimulus-evoked activity. It was hypothesized here that variations in ER-fMRI response amplitude evoked by successive stimuli may have reduced detection sensitivity in these previous studies. Multiple regression with orthogonal polynomials (OPR) was used to increase detection sensitivity by employing orthogonal polynomial equations to model such variations in response amplitude. ER-fMRI data was collected from 17 subjects during performance of the visual oddball task, which included frequent nontarget, rare nontarget distractor, and rare target block letter stimuli, to which subjects made a speeded button press response. When compared with multiple regression using main effect regressors alone, OPR identified an increased volume of significant target and distractor evoked main effect response due to reduced error variance. In addition, distractor-evoked activity was found to correlate with orthogonal polynomial regressors but not main effect regressors across a large volume of prefrontal and paralimbic brain regions including dorsal-lateral prefrontal, inferior-lateral prefrontal, and cingulate cortex. These results illustrate that considerable variability is present in ER-fMRI responses evoked by rare distractor stimuli in the oddball task. Such variability can be modeled using OPR, leading to increased detection sensitivity and better anatomical correspondence between the findings of ER-fMRI and ERP studies.

XY-TOF technique for large ion source mass spectrometers

The combination of time-of-flight (TOF) with two-dimensional position-sensitive ( XY) ion detection techniques is employed to increase detection sensitivity of gas mass spectrometers. For this, the length of ion sources is significantly increased without deteriorating the mass resolution; this is achieved by using the XY information to correct the TOFs of the ions produced in the collisions. The ion dynamics expressions for such XY-TOF method are deduced. The gas pressure may be kept at a uniform value all over the cell to facilitate accurate absolute measurements. Experimental results of 150 keV neutral H (H 0)-beam colliding with a He–Ne–Ar mixture are shown before and after the XY-TOF compensation.

Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model.

Mouse tumor models have undergone profound improvements in the fidelity of emulating human disease. Replacing ectopic s.c. implantation with organ-specific orthotopic implantation reproduces human tumor growth and metastasis. Strong fluorescent labeling with green fluorescent protein along with inexpensive video detectors, positioned externally to the mouse, allows the monitoring of details of tumor growth, angiogenesis, and metastatic spread. However, the sensitivity of external imaging is limited by light scattering in intervening tissue, most especially in skin. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold. The observable depth of tissue is thereby greatly increased and many tumors that were previously hidden are now clearly observable. This report presents tumor images and related quantitative growth data previously impossible to obtain. Single tumor cells, expressing green fluorescent protein, were seeded on the brain image through a scalp skin-flap. Lung tumor microfoci representing a few cells are viewed through a skin-flap over the chest wall, while contralateral micrometastases were imaged through the corresponding skin-flap. Pancreatic tumors and their angiogenic microvessels were imaged by means of a peritoneal wall skin-flap. A skin-flap over the liver allowed imaging of physiologically relevant micrometastases originating in an orthotopically implanted tumor. Single tumor cells on the liver arising from intraportal injection also were detectable. Possible future technical developments are suggested by the image, through a lower-abdominal skin-flap, of an invasive prostate tumor expressing both red and green fluorescent proteins in separate colonies.

Analysis of the herbicides paraquat, diquat and difenzoquat in drinking water by micellar electrokinetic chromatography using sweeping and cation selective exhaustive injection

Optimum conditions for the determination of the herbicides paraquat, diquat and difenzoquat by micellar electrokinetic chromatography (MEKC) using sweeping and cation-selective exhaustive injection (CSEI) as on-line concentration methods were developed. Sodium dodecyl sulfate (80 m M) in 50 m M phosphate buffer (pH 2.5) with 20% acetonitrile was used as a background electrolyte for the methods studied. The limits of detection, based on a signal-to-noise ratio of 3:1, were about 2.6–5.1 mg l −1 in purified water when MEKC was applied for the standards. By using an on-line preconcentration method known as sweeping–MEKC, up to a 500-fold increase in detection sensitivity was obtained whereas up to a 50 000-fold increase for CSEI–sweeping–MEKC was achieved. The limits of detection using optimum CSEI–sweeping–MEKC were lower than 1 μg l −1 and the method was validated obtaining good reproducibility (relative standard deviation lower than 22%) and linearity. CSEI–sweeping–MEKC was successfully applied to the determination of the three herbicides in spiked tap water below the levels established by the US Environmental Protection Agency.

搬送波抑制回路を用いた高周波キャリア型薄膜磁界センサの交流磁界検出感度

A high-frequency carrier type thin-film magnetic field sensor was combined with a high-frequency carrier suppressing circuit to increase detection sensitivity. The carrier-suppressing circuit was designed and tuned to suppress the carrier, thus reducing the phase noise level of thermal noise (-174 dBm) at room temperature. The carrier was suppressed by about 60 dB, enabling detection of a very low signal from the sensor. As a result, high sensitivity of 7.2 × 10-6A/m (9 × 10-8 Oe) was demonstrated at 501 kHz.

Recent advances in ion chromatography suppressor improve anion separation and detection

The Alltech DS-Plus suppressor improves and simplifies anion analysis by suppressor-based ion chromatography (IC). In addition to suppressing the mobile phase and enhancing the analyte signal, the DS-Plus suppressor removes carbonic acid from the suppressor effluent when carbonate–hydrogencarbonate mobile phases are used, reducing the water dip that interferes with early eluting peaks, increasing detection sensitivity, and enabling carbonate–hydrogencarbonate gradients. The ability to operate with carbonate–hydrogencarbonate gradients places exceptional separating power in the mobile phase, forgoing the need for expensive columns with special selectivity. Continuous operation and solid-phase chemistry eliminates the need for external regenerants, switching valves, and multiple operating modes.

Recognition of multiple classes of hepatitis C antibodies increases detection sensitivity in oral fluid.

Paired serum-oral fluid samples from 127 hepatitis C virus (HCV)-positive and 31 HCV-negative patients were tested for the presence of anti-HCV using the Ortho HCV 3.0 ELISA. Using the immunoglobulin G (IgG)-specific detection antibody provided with the HCV 3.0 ELISA we attained 100% sensitivity and specificity with serum samples; however, sensitivity in oral fluid samples was only 81%. By modifying the HCV 3.0 ELISA to utilize a secondary antibody cocktail that recognizes not only IgG but IgA and IgM as well, we attained 100% specificity and sensitivity with oral fluid samples.

Anion selective exhaustive injection-sweep–micellar electrokinetic chromatography

A selective on-line sample concentration technique of anion selective exhaustive injection (ASEI)-sweep–micellar electrokinetic chromatography (MEKC) was evaluated using a cationic surfactant. To suppress the electroosmotic flow, a polyacrylamide-coated capillary was introduced. Some aromatic carboxylic acids, dansyl amino acids, and naphthalenedisulfonic acids were used as test analytes. About 1000- to 6000-fold increases in detection sensitivity were obtained in terms of peak heights by ASEI-sweep–MEKC.

A capillary-based supersonic jet inlet system for resonance-enhanced laser ionization mass spectrometry: principle and first on-line process analytical applications.

A new supersonic jet inlet system for resonance-enhanced multiphoton ionization time-of-flight mass spectrometry (REMPI-TOFMS), based on a fused-silica capillary with an integral nozzle has been developed. The new jet inlet system generates a supersonic molecular beam that originates in the center of the ion source of the time-of-flight mass spectrometer. Because of the design of the inlet system, high spatial overlap of sample and laser beam (i.e., increased detection sensitivity) and excellent jet beam qualities are achieved with good adiabatic cooling properties of analyte molecules (i.e., considerably enhanced optical selectivity of the REMPI process). Furthermore, the inlet is very robust and chemically inert and contains no moving parts. As a result of these properties, the new inlet is perfectly suited for field applications of jet-REMPI. A first field application of a mobile supersonic jet-REMPI mass spectrometer equipped with the novel inlet technique is reported; namely, the concentration of monochlorobenzene, which is an indicator for the formation and emission of toxic polychlorinated dibenzo-p-dioxins/furans, PCDD/F) was measured on-line in the flue gas of a waste incineration plant.