Immunological Biosensor for Detection of Vibrio cholerae O1in Environmental Water Samples

Environmental surveillance for the presence of Vibrio cholerae O1 is of utmost importance for the effective public health protection of cholera. In the present study, an amperometric immunosensor was developed for detection of Vibrio cholerae in environmental samples by using disposable screen-printed electrodes (SPEs). For this purpose, the experiments done include fabrication of SPEs by using carbon ink, electrochemical characterization of electrodes, optimization of dilutions of antibodies and immobilization of antibody. V. cholerae O1 bacteria were spiked in various environmental water samples in known number. The seeded samples were filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly for detection of V. cholerae using the immunosensor. The immunosensor could detect as few as 8 c.f.u./ml in hand-pump water (ground water) and seawater, and 80 c.f.u./ml in sewer water and tap water. The total time taken in this detection assay was 55 min. Thus, the proposed method is simple and can be used for environmental monitoring of V.␣cholerae.     

Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay

Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5′ and 3′ ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 10⁴. A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.Clinical diagnostics and disease management strategies increasingly require fast and accurate methods for the detection and identification of multiple pathogenic microorganisms from complex samples. Conventional techniques used to detect and identify pathogenic microorganisms have typically relied upon culture-based morphological approaches. Unfortunately, these methods are often time-consuming, laborious, and restricted to those microorganisms that can be cultured routinely.Several recently developed molecular techniques, such as conventional and real-time PCRs, circumvent some of these drawbacks. PCR-based detection mechanisms are sensitive, accurate, and relatively fast and allow the detection of difficult-to-culture microorganisms. This last aspect is of considerable importance, given the fact that the majority of the microorganisms present in environmental samples still elude conventional cultivation efforts (1, 21). Although PCR-based methods for microbial identification and detection offer several advantages over conventional microbiological approaches, they still often have serious limitations. The attainable level of multiplexing is relatively low and is typically restricted to the detection of only a few target pathogens per assay (15, 40). Adding multiple specific primer pairs to a single reaction mixture can result in undesired amplification products (16), and for TaqMan PCR, the attainable level of multiplexing is low due to the limited number of fluorescent probes (8, 34, 41). Reliable detection and identification of several pathogens in a single sample, therefore, requires separate reaction mixtures, making large-scale screening of samples more laborious, time-consuming, and expensive. To increase efficiency and reduce expenses, it is desirable to develop simple and rapid multiplex assays that can specifically detect and identify several pathogens simultaneously.DNA microarray- and macroarray-based technologies offer the possibility of adding a highly multiplexed aspect to PCR-based pathogen detection and identification (9, 37, 54, 58). Array-based pathogen detection strategies typically involve PCR amplification of universal phylogenetic target genes (e.g., 16S, 18S, and 23S rRNA genes) or a number of microorganism-specific genetic markers (10, 19, 58) or random amplification of genomic DNA (gDNA) fragments (54). The combination of nucleic acid amplification strategies with array-based detection has resulted in the development of sensitive, high-throughput microbial diagnostic microarrays (MDMs) (4, 38, 50, 60). Although several array-based detection technologies have been realized to detect pathogens, only a minority of these methods can discriminate target pathogens from closely related nontarget organisms, which may differ from the target organisms by only a single nucleotide in the probe-binding region (36). Designing sufficiently discriminating oligonucleotide detectors for arrays, however, is relatively complicated, requiring extensive hybridization specificity testing. Moreover, the oligonucleotide detectors spotted onto the microarray are target organism specific, making it necessary to redesign microarrays if accommodation of additional probes is required for the detection of new targets.DNA ligase requires a double-stranded match to allow ligation, facilitating the development of ligation-based systems to discriminate point mutations (29). This feature of ligation detection (LD) has led to the development of several strategies for genotyping single-nucleotide polymorphisms (SNPs) and detecting pathogens (6, 7, 11, 46). However, current LD assays require two adjacent detection oligonucleotide probes with the same melting temperature (T_m) for each target sequence, although the use of intramolecular ligation, as in padlock probe (PLP) technologies, has been demonstrated to hold clear advantages (44, 45, 51). PLPs are long oligonucleotides, ∼100 bases long, containing target complementary arms at both termini of the probe. In the assay developed in this study, the target complementary arms are connected via a compound linker sequence containing spacer sequences, a thymine-linked desthiobiotin moiety for specific capture and release (25, 53), deoxyuracil nucleotides for probe cleavage, and a unique sequence identifier, the so-called ZipCode, for standardized microarray hybridization (18) (Fig. ​(Fig.1A).1A). The unimolecular nature of the PLP allows asymmetric target complementary arm design, whereby a long 5′ arm serves as an anchor sequence and the short 3′ arm, with a low T_m, facilitates extremely specific target detection (17, 51, 53). Microarray-based PLP technology was previously shown to provide reliable detection of multiple pathogenic microorganisms, but PCR amplification of residual, unligated PLPs resulted in significant background signals, thereby complicating data analysis and decreasing the overall dynamic range of reliable detection (3, 51).Open in a separate windowFIG. 1.Schematic overview of the novel single-molecule LD system. (A) PLP design. T1a and T1b are asymmetric target complementary regions. Each PLP contains a unique ZipCode sequence for universal array hybridization, two spacer sequences (S1 and S2), a desthiobiotin moiety (dBio) for probe capture, a polyoligo(dT) linker sequence, and a polydeoxyuracil sequence for probe cleavage. (B) Multiple target-specific PLPs are ligated to PCR-preamplified DNA samples. T1a and T1b bind to adjacent sequences of the target, and in the case of a perfect match, the probe is circularized by enzymatic ligation. The PLPs are reversibly captured and washed via the desthiobiotin moiety with magnetic streptavidin-coated beads. Next, the washed probes are cleaved at the polydeoxyuracil sequences with UNG and endonuclease enzymes. The sample containing the cleaved PLPs is hybridized on a universal microarray. Finally, only the hybridized PLPs that were originally ligated can be labeled and visualized with streptavidin R-PE by using the desthiobiotin moiety.Here, we describe the development, testing, and implementation of a novel, background-free, LD-dependent strategy in which multiple PLPs are ligated on fragmented, PCR-preamplified DNA sequences. The target complementary regions recognize adjacent sequences on the target DNA, and ligation occurs only if the end nucleotides perfectly match their target, resulting in a circular molecule (Fig. ​(Fig.1B).1B). Next, the probes are captured with streptavidin-coupled magnetic beads, allowing separation from the rest of the sample. Subsequently, the washed probes are eluted from the beads and cut at the internal polydeoxyuracil probe region by enzymatic cleavage. Thus, the desthiobiotin moiety needed for fluorescent labeling of unligated PLPs is removed, while the ligated probes are linearized (Fig. ​(Fig.1B).1B). Finally, the sample is hybridized on a universal complementary ZipCode (cZipCode) microarray (18) and visualized via fluorescent labeling of the desthiobiotin moiety (Fig. ​(Fig.1B1B).In this paper, we report the development and application of cleavable PLPs combined with LD for the simultaneous, background-free detection and identification of multiple plant pathogens in environmental samples. The specificity, sensitivity, and dynamic range of detection of the developed assay were determined by using nine target-specific PLPs, and the robustness of the assay was evaluated by using samples collected from hydroponic horticultural water recirculation systems.     

Detection of Microcystin-Producing Cyanobacteria in Finnish Lakes with Genus-Specific Microcystin Synthetase Gene E (mcyE) PCR and Associations with Environmental Factors

We studied the frequency and composition of potential microcystin (MC) producers in 70 Finnish lakes with general and genus-specific microcystin synthetase gene E (mcyE) PCR. Potential MC-producing Microcystis, Planktothrixand Anabaena spp. existed in 70%, 63%, and 37% of the lake samples, respectively. Approximately two-thirds of the lake samples contained one or two potential MC producers, while all three genera existed in 24% of the samples. In oligotrophic lakes, the occurrence of only one MC producer was most common. The combination of Microcystis and Planktothrix was slightly more prevalent than others in mesotrophic lakes, and the cooccurrence of all three MC producers was most widespread in both eutrophic and hypertrophic lakes. The proportion of the three-producer lakes increased with the trophic status of the lakes. In correlation analysis, the presence of multiple MC-producing genera was associated with higher cyanobacterial and phytoplankton biomass, pH, chlorophyll a, total nitrogen, and MC concentrations. Total nitrogen, pH, and the surface area of the lake predicted the occurrence probability of mcyE genes, whereas total phosphorus alone accounted for MC concentrations in the samples by logistic and linear regression analyses. In conclusion, the results suggested that eutrophication increased the cooccurrence of potentially MC-producing cyanobacterial genera, raising the risk of toxic-bloom formation.     

Detection of Cytomegalovirus Antibodies Using a Biosensor Based on Imaging Ellipsometry

Background

Cytomegalovirus (CMV) is the most common infectious cause of mental disability in newborns in developed countries. There is an urgent need to establish an early detection and high-throughput screening method for CMV infection using portable detection devices.

Methods

An antibody analysis method is reported for the detection and identification of CMV antibodies in serum using a biosensor based on high spatial resolution imaging ellipsometry (BIE). CMV antigen (CMV-3A) was immobilized on silicon wafers and used to capture CMV antibodies in serum. An antibody against human immunoglobulin G (anti-IgG) was used to confirm the IgG antibody against CMV captured by the CMV-3A.

Results

Our results show that this assay is rapid and specific for the identification of IgG antibody against CMV. Further, patient serum was quantitatively assessed using the standard curve method, and the quantitative results were in agreement with the enzyme-linked immunosorbent assay. The CMV antibody detection sensitivity of BIE reached 0.01 IU/mL.

Conclusions

This novel biosensor may be a valuable diagnostic tool for analysis of IgG antibody against CMV during CMV infection screening.     

Detection of Diverse New Francisella-Like Bacteria in Environmental Samples

Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.     

Carbon Concentration Mechanism(s) in Unicellular Green Algae and Cyanobacteria

Unicellular green algae and cyanobacteria have mechanism(s) to actively concentrate dissolved inorganic carbon (DIC) into the cells, only if they are grown with air levels of CO₂. The DIC concentration mechanisms are environmental adaptations to actively transport and accumulate inorganic carbon into the chloroplasts of green algae or into the carboxysomes of cyanobacteria. The current working model of cyanobacterial carbon concentration mechanism consists of at least two basic components: an active C_i transport system and a Rubisco-rich polyhedral carboxysome. In case of unicellular green algae, the working model for DIC concentration mechanism includes several isoforms of carbonic anhydrase (CA), and ATPase driven active bicarbonate transporters at the plasmalemma and at the inner chloroplast envelopes. In the past twenty years, significant progress has been made in isolating and characterizing the isoforms of carbonic anhydrase. However, active transporters are yet to be characterized. This mini-review summarizes the current status of research on DIC-pumps including its significance and possible application to increase the productivity of plants of economic importance.     

Detection of Protein A Produced by Staphylococcus aureus with a Fiber-optic-based Biosensor

Staphylococcus aureus is a pathogen important in causing human infections and intoxication. A sensitive fiber-optic that produces evanescent waves was developed for the detection of protein A, a product secreted only by S. aureus. In the immunosensor, a 40-mV argon-ion laser that generated laser light at 488 nm was used together with plastic optical fiber and antibodies to protein A were physically adsorbed onto the fiber. The principle of the detection involved a sandwich immunoassay with fluorescein isothiocyanate conjugated with anti-(protein A) immunoglobulin G to produce signals of the antigen-antibody reaction. The detection limit was 1 ng of protein A per milliliter. The fiber-optic immunosensor could be used for rapid and specific detection of S. aureus in clinical specimens and foods.     

Detection of Protozoan Hosts for Legionella pneumophila in Engineered Water Systems by Using a Biofilm Batch Test

Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.Legionella pneumophila, the causative agent of Legionnaires'' disease, is a common inhabitant of natural freshwater environments and human-made water systems, including cooling towers, whirlpools, air-conditioning systems, and installations for warm tap water (14). In the aquatic environment L. pneumophila proliferates within certain free-living protozoa, which serve as its hosts (15, 30, 59). Environmental factors favoring the growth and survival of L. pneumophila in freshwater systems include a water temperature between 20°C and 45°C (41, 60) and the presence of biofilms and sediments on which the protozoan hosts can graze (30, 41, 56).Rowbotham (44) was the first to report the growth of L. pneumophila within free-living amoebae, which belonged to the genera Acanthamoeba and Naegleria. In vitro studies with cocultures have revealed that 14 species of amoebae, viz., Acanthamoeba spp. (1, 35, 44, 53), Balamuthia mandrillaris (47), Echinamoeba exundans (15), Hartmannella spp. (43), Naegleria spp. (38, 44, 53), and Vahlkampfia jugosa (43); the slime mold Dictyostelium discoideum (20, 48); and two species of the ciliate genus Tetrahymena (15, 26) can serve as hosts for L. pneumophila. Recently, it has been reported that L. pneumophila can also replicate within the intestinal tract of the microbiovorous nematode Caenorhabditis elegans (3).A number of the free-living protozoa mentioned above and others, e.g., Vannella spp. and Saccamoeba spp., have been observed in aquatic environments from which L. pneumophila was cultivated or in which it was detected with PCR (4, 42, 51, 52). However, it remains unknown which of these protozoa actually serve as hosts for L. pneumophila in the aquatic environment, including human-made water systems. Moreover, it cannot be excluded that free-living protozoa other than those tested in vitro can serve as hosts for L. pneumophila as well. Information is also lacking about protozoan hosts for Legionella anisa (13, 49), which is frequently present in water installations in temperate regions (11, 62). Furthermore, it is unknown which free-living protozoa serve as hosts for uncultured Legionella bacteria that can grow at temperatures of about 15°C (61; B. A. Wullings, G. Bakker, and D. van der Kooij, submitted for publication).L. pneumophila can proliferate in samples of surface water, effluent of wastewater treatment plants, potable water, and water from cooling towers incubated at 25°C, 35°C, or 37°C (28, 45, 56). Consequently, incubation of freshwater samples can be used to amplify protozoan hosts for L. pneumophila and other Legionella spp. In this study, different human-made water types were investigated using a biofilm batch test (BBT) system to (i) amplify and subsequently identify predominating, known, and yet-undescribed hosts for L. pneumophila and (ii) identify potential protozoan hosts for Legionella bacteria that can grow at 15°C.     

Detection and Enumeration of a Tagged Pseudomonas fluorescens Strain by Using Soil with Markers Associated with an Engineered Catabolic Pathway

Previously we described a novel gene tagging method, using the moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955, to identify microorganisms destined for release into the environment. Here, we used the engineered strain Pseudomonas fluorescens PF5MT12 carrying the moc region integrated into the bacterial chromosome to demonstrate the usefulness of the markers for detection and direct selection of marked organisms present in soil samples. Using this system, we routinely detected population levels as low as 10(sup2) CFU per g of soil sampled. In addition to direct selection, we developed an immunologically based assay using MOP cyclase, a unique enzyme associated with moc, as the epitope for detecting the tagged organism. The colony immunoblot assay proved to be highly specific and without any false-positive signals when used to identify organisms cultured from soil on nonselective medium. The numbers of colonies that were immunoreactive with the anti-MOP cyclase antibody were essentially equal to those that grew out on selection plates. This indicates that MOP cyclase can be used as a marker and that we can use nonselective medium to retrieve the marked genetically engineered microorganisms and then identify them by using colony immunoblot assays. These direct selection and colony immunoblot methods provide a sensitive and accurate strategy for identifying and enumerating marked organisms recovered from soil samples. We also developed a rapid assay for MOP cyclase that does not require cell permeabilization with toluene. This assay can be used to verify tagged organisms isolated by other methods or to screen large numbers of colonies for the tag following nonselective isolation.     

Biotransformation of Hg(II) by Cyanobacteria

The biotransformation of Hg(II) by cyanobacteria was investigated under aerobic and pH-controlled culture conditions. Mercury was supplied as HgCl₂ in amounts emulating those found under heavily impacted environmental conditions where bioremediation would be appropriate. The analytical procedures used to measure mercury within the culture solution, including that in the cyanobacterial cells, used reduction under both acid and alkaline conditions in the presence of SnCl₂. Acid reduction detected free Hg(II) ions and its complexes, whereas alkaline reduction revealed that meta-cinnabar (β-HgS) constituted the major biotransformed and cellularly associated mercury pool. This was true for all investigated species of cyanobacteria: Limnothrix planctonica (Lemm.), Synechococcus leopoldiensis (Racib.) Komarek, and Phormidium limnetica (Lemm.). From the outset of mercury exposure, there was rapid synthesis of β-HgS and Hg(0); however, the production rate for the latter decreased quickly. Inhibitory studies using dimethylfumarate and iodoacetamide to modify intra- and extracellular thiols, respectively, revealed that the former thiol pool was required for the conversion of Hg(II) into β-HgS. In addition, increasing the temperature enhanced the amount of β-HgS produced, with a concomitant decrease in Hg(0) volatilization. These findings suggest that in the environment, cyanobacteria at the air-water interface could act to convert substantial amounts of Hg(II) into β-HgS. Furthermore, the efficiency of conversion into β-HgS by cyanobacteria may lead to the development of applications in the bioremediation of mercury.     

Detection and Enumeration of a Tagged Pseudomonas fluorescens Strain from Soil by Using Markers Associated with an Engineered Catabolic Pathway

Volume 63, no. 2, p. 602: the article title should read as shown above. [This corrects the article on p. 602 in vol. 63.].     

Using a Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Chicken Carcass

Chicken is one of the most popular meat products in the world. Salmonella Typhimurium is a common foodbome pathogens associated with the processing of poultry. An optical Surface Plasmon Resonance （SPR） biosensor was sensitive to the presence of Salmonella Typhimurium in chicken carcass. The Spreeta biosensor kits were used to detect Salmonella Typhimurium on chicken carcass successfully. A taste sensor like electronic tongue or biosensors was used to basically ＂taste＂ the object and differentiated one object from the other with different taste sensor signatures. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with dif- ferent antisera or other molecular species. The selectivity of the SPR biosensor was assayed using a series of antibody con- centrations and dilution series of the organism. The SPR biosensor showed promising to detect the existence of Salmonella Typhimurium at 1 x 106 CFU/ml. Initial results show that the SPR biosensor has the potential for its application in pathogenic bacteria monitoring. However, more tests need to be done to confirm the detection limitation.     

Development of a Biosensor for Detection of Pleural Mesothelioma Cancer Biomarker Using Surface Imprinting

Hyaluronan-linked protein 1 (HAPLN1) which has been shown to be highly expressed in malignant pleural mesotheliomas (MPM), was detected in serum using an electrochemical surface-imprinting method. First, the detection method was optimized using Bovine serum albumin (BSA) as a model protein to mimic the optimal conditions required to imprint the similar molecular weight protein HAPLN1. BSA was imprinted on the gold electrode with hydroxyl terminated alkane thiols, which formed a self-assembled monolayer (SAM) around BSA. The analyte (BSA) was then washed away and its imprint (empty cavity with shape-memory) was used for detection of BSA in a solution, using electrochemical open-circuit potential method, namely potentiometry. Factors considered to optimize the conditions include incubation time, protein concentration, limit of detection and size of electrode. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to confirm selectivity of imprints. With the obtained imprinting control parameters, HAPLN1 was imprinted in duplicate and the detection of spiked HAPLN1 was successfully conducted in serum.     

Detection of anatoxin-a(s) in environmental samples of cyanobacteria by using a biosensor with engineered acetylcholinesterases

Bioassays are little used to detect individual toxins in the environment because, compared to analytical methods, these assays are still limited by several problems, such as the sensitivity and specificity of detection. We tentatively solved these two drawbacks for detection of anatoxin-a(s) by engineering an acetylcholinesterase to increase its sensitivity and by using a combination of mutants to obtain increased analyte specificity. Anatoxin-a(s), a neurotoxin produced by some freshwater cyanobacteria, was detected by measuring the inhibition of acetylcholinesterase activity. By using mutated enzyme, the sensitivity of detection was brought to below the nanomole-per-liter level. However, anatoxin-a(s) is an organophosphorous compound, as are several synthetic molecules which are widely used as insecticides. The mode of action of these compounds is via inhibition of acetylcholinesterase, which makes the biotest nonspecific. The use of a four-mutant set of acetylcholinesterase variants, two mutants that are sensitive to anatoxin-a(s) and two mutants that are sensitive to the insecticides, allows specific detection of the cyanobacterial neurotoxin.     

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

Genus- and Species-Specific PCR-Based Detection of Dairy Propionibacteria in Environmental Samples by Using Primers Targeted to the Genes Encoding 16S rRNA

PCR assays with primers targeted to the genes encoding 16S rRNA were developed for detection of dairy propionibacteria. Propionibacterium thoenii specific oligonucleotide PT3 was selected after partial resequencing. Tests allowed the detection of less than 10 cells per reaction from milk and cheese and 10² cells per reaction from forage and soil.     

Design of a Biosensor for the Detection of Dengue Virus Using 1D Photonic Crystals

Plasmonics - A biosensor for the detection of dengue virus has been designed using 1D photonic crystal. In the proposed structure [(Si/LiF)6D(LiF/Si)6], D is the defect layer. Refractive index of...     

PCR-Based Ribosomal DNA Detection Technique for Microalga (Heterosigma carterae) Causing Red Tide and Its Application to a Biosensor Using Labeled Probe

A technique for detecting Raphidophycean, a bloom-forming genus of algae, was developed using a specific DNA probe. The design of the probe was based on a sequence polymorphism within the small subunit (SSU) ribosomal RNA gene (rDNA) of this strain by using fluorescence polarization (FP) analysis and the BIAcore 2000 biosensor, which utilized surface plasmon resonance (SPR). The specific sequence in SSU rDNA for Heterosigma carterae was determined by sequence data analysis. One pair of polymerase chain reaction (PCR) probes was designed for use in making the identification. H. carterae SSU rDNA was amplified by PCR. Using a fluoroscein isothiocyanate–labeled or biotin-labeled oligonucleotide probe, the PCR-amplified rDNA was selectively detected as an FP-intensity change via FP analysis or as a resonance-unit change via SPR. Although total time for final detection after sampling was within 3 hours, specific rDNA could be detected within 10 minutes after PCR through these detection methods.     

First Detection of Mycobacterium ulcerans DNA in Environmental Samples from South America

The occurrences of many environmentally-persistent and zoonotic infections are driven by ecosystem changes, which in turn are underpinned by land-use modifications that alter the governance of pathogen, biodiversity and human interactions. Our current understanding of these ecological changes on disease emergence however remains limited. Buruli ulcer is an emerging human skin disease caused by the mycobacterium, Mycobacterium ulcerans, for which the exact route of infection remains unclear. It can have a devastating impact on its human host, causing extensive necrosis of the skin and underlying tissue, often leading to permanent disability. The mycobacterium is associated with tropical aquatic environments and incidences of the disease are significantly higher on floodplains and where there is an increase of human aquatic activities. Although the disease has been previously diagnosed in South America, until now the presence of M. ulcerans DNA in the wild has only been identified in Australia where there have been significant outbreaks and in western and central regions of Africa where the disease is persistent. Here for the first time, we have identified the presence of the aetiological agent''s DNA in environmental samples from South America. The DNA was positively identified using Real-time Polymerase Chain Reaction (PCR) on 163 environmental samples, taken from 23 freshwater bodies in French Guiana (Southern America), using primers for both IS2404 and for the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes (KR). Five samples out of 163 were positive for both primers from three different water bodies. A further nine sites had low levels of IS2404 close to a standard CT of 35 and could potentially harbour M. ulcerans. The majority of our positive samples (8/14) came from filtered water. These results also reveal the Sinnamary River as a potential source of infection to humans.