Detection of two Prorocentrum species using sandwich hybridization integrated with nuclease protection assay

A novel assay method using nuclease protection assay integrated with sandwich hybridization (NPA-SH) for qualitative and quantitative detection of microalgae has been developed. Two species-specific nuclease-protection-assay (NPA) probes targeted 28S ribosomal RNA of Prorocentrum minimum and Prorocentrum micans, respectively, were designed in this study. The assay consists of S1 nuclease protection, sandwich hybridization and signal detection. The specificity of the probes was verified with cultured algae in the laboratory and field sample from Jiaozhou Bay, and the quantity by NPA-SH analysis showed good agreement with that of cell-counting with a light microscope. The optical absorbance of probe binding on the target showed good linear fit with cell amount. A standard curve for P. minimum was established to correlate the optical absorbance to cell density on a basis in the linear range between 15 and 475 cells ml⁻¹ seawater, and the equation deducted was ‘y = 0.0053 × x + 0.0658’ (R² = 0.992, n = 4). The assay was sensitive to detect 15 cells ml⁻¹ seawater. And for P. micans, with linear range between 0.6 and 20 cells ml⁻¹ seawater, the equation deducted was ‘y = 0.1174 × x + 0.1106’ (R² = 0.996, n = 4); the assay was sensitive to detect less than 1 cell ml⁻¹ seawater. The inter-assay coefficients of variation (CVs) were 12.4 and 10.9%, respectively. The good specificity, sensitivity and reproducibility of the NPA-SH implied that this new technique could be extremely useful for qualitative and quantitative assay of P. minimum and P. micans at low abundance.     

Ostracodology in time and space: looking back on fifteen International Symposia on Ostracoda,and the times in between

Nuclease protection assay (NPA) probes were designed to target the rRNA of Chaetoceros curvisetus and Skeletonema costatum, and quantitative sandwich hybridization integrated with nuclease protection assay (NPA-SH) was developed to detect C. curvisetus and S. costatum in culture and field samples in Jiaozhou Bay, China. The specificity and validity of the NPA-SH technique were tested with cultured pure strains, mixed strains and field samples, and by comparison with that of microscopy observation. The linear detection range for C. curvisetus was 4.2 × 10⁴ to 1.2 × 10⁶ cells with a detection limit of 42 cells ml⁻¹. The linear range for S. costatum was 6.0 × 10⁴ to 1.0 × 10⁶ cells with a detection limit of 60 cells ml⁻¹. The NPA-SH in this study provides a convenient tool for rapid assessment of HAB species in marine environments. Handling editor: D. Hamilton     

Species-Specific Detection and Quantification of Toxic Marine Dinoflagellates Alexandrium tamarense and A. catenella byReal-Time PCR Assay

A Real-time polymerase chain reaction (PCR) assay was designed and evaluated for rapid detection and quantification of the toxic dinoflagellates Alexandrium catenella and A. tamarense, which cause paralytic shellfish poisoning. Two sets of PCR primers and fluorogenic probes targeting these two species were derived from the sequence of 28S ribosomal DNA. PCR specificity was examined in closely related Alexandrium spp. and many other microalgae. A. catenella–specific primers and probe detected the PCR amplification only from A. catenella strains, and nonspecific signals were not detected from any microalgae. Also, A. tamarense–specific primers and probe also detected the targeted species, suggesting the strict species specificity of each PCR. This assay could detect one cell of each species, showing its high sensitivity. Moreover, using the developed standard curves, A. tamarense and A. catenella could be quantified in agreement with the quantification by optical microscopy. The performance characteristics of species specificity, sensitivity, and rapidity suggest that this method is applicable to the monitoring of the toxic A. tamarense and A. catenella.     

Identification and quantification of the toxic dinoflagellate Gymnodinium sp. with competitive enzyme-linked immunosorbent assay (cELISA)

Polyclonal antibodies were raised against Gymnodinium sp. by immunizing rabbits with cells of the axenic strain. Based on the species-specific antiserum, an indirect competitive enzyme-linked immunosorbent assay (cELISA) was developed to identify and quantify Gymnodinium sp. A standard curve was established to correlate the cELISA signal to cell amount on a logit-log basis in the linear range between 24 and 6,250,000 cells, and the equation deducted was ln[A/(A₀ − A)]= 4.9193 − 1.1006 log[cell amount] (R² = 0.9948, n = 5). The detection limit was found to be 12 cells. The intra-assay and inter-assay coefficients of variation (CVs) were 5.8% and 9.7%, respectively. Field samples collected from Jiaozhou Bay, China were used to assess the robustness of the method. The results showed high agreement with that of cell-counting with a light microscope. The good reproducibility and precision of the cELISA implied that this new technique could be used for fast quantification of Gymnodinium sp.     

Colorimetric detection of the toxic dinoflagellate Alexandrium minutum using sandwich hybridization in a microtiter plate assay

Rapid and reliable detection of harmful algae in coastal areas and shellfish farms is an important requirement for monitoring programs. Molecular technologies are rapidly improving the detection of phytoplankton and their toxins. Assays are based on the discrimination of genetic differences in the species. A commercially available PCR ELISA Dig Detection Kit in a microtiter plate was adapted for the rapid assessment of specificity of the two probes used in a sandwich hybridization assay. The toxic dinoflagellate Alexandrium minutum was used as the target organism and a capture and signal probe were designed for a species-specific identification of this species. This assay also provided the necessary specificity tests prior to the probes being adapted to an automated biosensor using a sandwich hybridization format. All probes regardless of the detection method must be extensively tested prior to use in the field. Total rRNA was isolated from three different strains of A. minutum and the mean concentration of RNA per cell of was determined to be 0.028 ng ± 0.003. Thus, a standard calibration curve for different RNA concentrations was determined so that cell numbers could be inferred from the assay. The assay and the standard curve were evaluated by using spiked field samples. The results demonstrated that the molecular assay was able to detect A. minutum cells at different cell counts in the presence of a complex background.     

Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments

The dinoflagellates Alexandrium tamarense (Lebor) Balech and Alexandrium catenella (Whedon and Kofoid) Balech (Dinophyceae) are believed to be the main species responsible for paralytic shellfish poisoning (PSP) all over the world. It is necessary to identify A. tamarense and A. catenella cysts and to monitor their distribution in sediment in order to minimize the damages caused by PSP to the economy and food quality because cysts are the seed population for blooms caused by motile vegetative cells. In this study, we developed an efficient DNA extraction method from the natural cysts present in marine sediments after they were size fractionated with a plankton net (mesh size of 20–150 μm). The 10–3000 cysts were added to the sediments collected from the Ariake Sea, and for which the primuline-staining method did not reveal any cysts. DNA was then extracted from each sample, and linear standard curves for A. tamarense and A. catenella cysts were obtained from the correlation between the Ct values by real-time PCR and the log of the initial densities of cysts. We monitored the A. tamarense and A. catenella cyst densities in the environmental samples. This assay was demonstrated to be a powerful tool for the identification, detection, and quantification of the cysts of the toxic dinoflagellates.     

Toxin profile of Alexandrium catenella from the Chilean coast as determined by liquid chromatography with fluorescence detection and liquid chromatography coupled with tandem mass spectrometry

The profile of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP) was determined from a Chilean strain of the marine dinoflagellate Alexandrium catenella. The toxin composition was compared with that of toxic shellfish, presumably contaminated by natural blooms of A. catenella from the same region in southern Chile. Ion pair-liquid chromatography with post-column derivatization and fluorescence detection (LC-FD) was employed for relative quantitative analysis of the toxin components, whereas unambiguous identification of the toxins was confirmed by tandem mass spectrometry (LC–MS/MS). In the dinoflagellate strain from Chile, the N-sulfocarbamoyl derivatives (C1/C2, B1) and the carbamoyl gonyautoxins GTX1/GTX4 comprise >90% of the total PSP toxin content on a molar basis. This toxin composition is consistent with that determined for A. catenella populations from the Pacific coast in the northern hemisphere. The characteristic toxin profile is also reflected in the shellfish, but with evidence of epimerization and metabolic transformations of C1 and C2 to GTX2 and GTX3, respectively. This work represents the first unequivocal identification and confirmation of such PSP toxin components from the Chilean coast.     

Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei,a Tier 1 select agent

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/μL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.     

Parallel Analyses of Alexandrium catenella Cell Concentrations and Shellfish Toxicity in the Puget Sound

Alexandrium catenella is widespread in western North America and produces a suite of potent neurotoxins that cause paralytic shellfish poisoning (PSP) in humans and have deleterious impacts on public health and economic resources. There are seasonal PSP-related closures of recreational and commercial shellfisheries in the Puget Sound, but the factors that influence cell distribution, abundance, and relationship to paralytic shellfish toxins (PSTs) in this system are poorly described. Here, a quantitative PCR assay was used to detect A. catenella cells in parallel with state shellfish toxicity testing during the 2006 bloom season at 41 sites from April through October. Over 500,000 A. catenella cells liter⁻¹ were detected at several stations, with two main pulses of cells driving cell distribution, one in June and the other in August. PSTs over the closure limit of 80 μg of PST 100 per g of shellfish tissue were detected at 26 of the 41 sites. Comparison of cell numbers and PST data shows that shellfish toxicity is preceded by an increase in A. catenella cells in 71% of cases. However, cells were also observed in the absence of PSTs in shellfish, highlighting the complex relationship between A. catenella and the resulting shellfish toxicity. These data provide important information on the dynamics of A. catenella cells in the Puget Sound and are a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this system.Various species of the dinoflagellate genus Alexandrium, including members of the species complex comprising Alexandrium catenella, Alexandrium fundyense, and Alexandrium tamarense, produce saxitoxins and a number of related derivatives (1). Shellfish that ingest toxic Alexandrium cells accumulate these potent neurotoxins, which can then lead to paralytic shellfish poisoning (PSP) in human consumers of shellfish. As such, paralytic shellfish toxins (PSTs) pose a serious threat to both public health and economically important fisheries (16). Within the Alexandrium genus, A. catenella is widespread in the northwestern part of North America, including the Puget Sound, and is responsible for seasonal harmful algal blooms (HABs) in this region (17). In the Puget Sound, recreational shellfish harvesters collect nearly 2 million pounds of clams and oysters annually, and Washington is also a leading producer of farmed bivalve shellfish in the United States, generating an estimated $77 million in sales a year and supporting thousands of jobs (13).PSTs are not a new problem in the Pacific Northwest; events have been documented as far back as the late 18th century (17). Currently, the Sentinel Monitoring Program of the Washington State Department of Health (WADOH) is in place to provide systematic early warning of harmful levels of PSTs, with caged mussels sampled at as many as 70 sites throughout all basins of Puget Sound at roughly 2-week intervals. Analysis of this long-term shellfish monitoring data indicates that maximum PST levels and PST-related closures have increased over the past 20 years, reaching >10,000 μg of PST per 100 g of shellfish tissue in multiple years and resulting in significant negative impacts on shellfisheries in the region (17).To date, monitoring efforts in the Puget Sound have focused on measuring the level of PSTs present in shellfish tissue. Existing programs do not typically monitor for phytoplankton species composition or abundance. Information on A. catenella distribution and seasonal dynamics is limited for this region, despite its potential value for monitoring and understanding toxic A. catenella blooms and their impacts. Toward this end, we used a previously developed high-throughput quantitative PCR (qPCR) method (5, 6) to detect and enumerate A. catenella cells. We couple this specific and sensitive detection method for A. catenella with PST monitoring efforts to examine changes in A. catenella populations and accompanying shellfish toxicity in the Puget Sound. The data, collected from April through October, span nearly all of the 2006 A. catenella bloom season in the region. These results provide important information on the abundance and dynamics (e.g., possible source populations) of A. catenella cells during a bloom season and on their relationship to PSTs in shellfish. This effort represents a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this region.     

Dot-immunobinding assay in the detection of IgG antibodies against farmer's lung antigens

Circulating antibodies against Faenia rectivirgula, Thermoactinomyces candidus, T. vulgaris and Aspergillus fumigatus were studied in the sera of 14 clinically proven farmer's lung patients and 10 normal controls using three immunological methods. These methods were agar gel double diffusion (DD), biotin-avidin-linked immunosorbent assay (BALISA) and dot-immunobinding assay (DIBA). Agar gel diffusion, the least sensitive of the three methods, failed to detect antibodies in some of the patients, while BALISA detected antibodies even in the normal controls. However, the sensitivity of dot-immunobinding assay was in between DD and BALISA while the specificity was comparable to DD to all the antibodies except against A. fumigatus antigens. Dot-immunobinding assay gave faster results than DD and the blots can be stored as record for longer periods of time without fading.     

Examination of the seasonal dynamics of the toxic dinoflagellate Alexandrium catenella at Redondo Beach, California, by quantitative PCR

The presence of neurotoxic species within the genus Alexandrium along the U.S. coastline has raised concern of potential poisoning through the consumption of contaminated seafood. Paralytic shellfish toxins (PSTs) detected in shellfish provide evidence that these harmful events have increased in frequency and severity along the California coast during the past 25 years, but the timing and location of these occurrences have been highly variable. We conducted a 4-year survey in King Harbor, CA, to investigate the seasonal dynamics of Alexandrium catenella and the presence of a particulate saxitoxin (STX), the parent compound of the PSTs. A quantitative PCR (qPCR) assay was developed for quantifying A. catenella in environmental microbial assemblages. This approach allowed for the detection of abundances as low as 12 cells liter⁻¹, 2 orders of magnitude below threshold abundances that can impact food webs. A. catenella was found repeatedly during the study, particularly in spring, when cells were detected in 38% of the samples (27 to 5,680 cells liter⁻¹). This peak in cell abundances was observed in 2006 and corresponded to a particulate STX concentration of 12 ng liter⁻¹, whereas the maximum STX concentration of 26 ng liter⁻¹ occurred in April 2008. Total cell abundances and toxin levels varied strongly throughout each year, but A. catenella was less abundant during summer, fall, and winter, when only 2 to 11% of the samples yielded positive qPCR results. The qPCR method developed here provides a useful tool for investigating the ecology of A. catenella at subbloom and bloom abundances.     

Colorimetric immuno-protein phosphatase inhibition assay for specific detection of microcystins and nodularins of cyanobacteria

A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and nodularin detection and analysis do not provide information on the toxicity of microcystin and/or nodularin variants. Furthermore, protein phosphatase inhibition-based assays for these toxins are not specific and respond to other environmental protein phosphatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We addressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a colorimetric protein phosphatase inhibition system in a single assay, designated the colorimetric immuno-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin variants (microcystin-LR, -D-Asp3-RR, -LA, -LF, -LY, -LW, and -YR) and nodularin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and nodularin-containing laboratory strains and environmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (R2 = 0.94, P < 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA procedure combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and nodularins.     

Rapid detection of natural cells of Alexandrium tamarense and A. catenella (Dinophyceae) by fluorescence in situ hybridization

The marine toxic dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor that cause paralytic shellfish poisoning (PSP) are identified on the basis of morphological features in routine monitoring. Rapid and simple identification is, however, often difficult because of the morphological similarity. Fluorescent in situ hybridization (FISH) using ribosomal RNA (rRNA)-targeted probes has been studied as a method of easily identifying and enumerating species responsible for harmful algal blooms (HABs). Its application to monitoring natural populations of HAB species, however, is limited. Here, we applied the FISH method to identify and enumerate cells of A. tamarense and A. catenella in natural plankton assemblages collected from Japanese coastal waters. A. tamarense-specific (Atm1) and A. catenella-specific (Act1) probes were established based on the D2 region of the large-subunit ribosomal RNA gene (28S rDNA). With these two probes, natural cells of A. tamarense or A. catenella in field samples could easily be identified when the following three conditions were met. First, cells should be concentrated by filtration, not centrifugation, in order to avoid the loss of cells. Second, autofluorescence should be minimized; acetone was an effective decolorization reagent. Third, samples should be stored at −20 or −80 °C for long-term preservation. The results indicate that FISH is a useful tool for the rapid identification of toxic Alexandrium spp. and can facilitate the analysis of numerous natural samples.     

Utility of pentose colorimetric assay for the purification of potato lectin, an arabinose-rich glycoprotein

Potato lectin (Solanum tuberosum agglutinin, STA) is an unusual glycoprotein containing approximately 50% carbohydrates by weight. Of the total carbohydrates, 92% is contributed by L-arabinose, which are O-linked to hydroxyproline residues. The ferric chloride-orcinol assay (Bial’s test), which is specific for pentoses has so far been used only for the determination of free pentoses in biological samples. However, this colorimetric assay has not been used for the detection of pentoses in bound form as it occurs in Solanaceae lectins (potato, tomato, and Datura lectins). Utilizing the pentose colorimetric assay for monitoring the presence of potato lectin, a simpler and shorter procedure for the purification of this lectin from potato tubers has been developed. The yield of potato lectin (1.73 mg per 100 g potato tuber) is twice compared to the yields reported in earlier procedures. Although potato lectin is well known for its specificity to free trimers and tetramers of N-acetyl-D-glucosamine (GlcNAc), it possesses a similar specificity to the core (GlcNAc)₂ of N-linked glycoproteins. The utilization of the pentose assay in the purification of arabinose-rich lectins/agglutinins obviates the necessity for the use of agglutination assay in the various purification steps. The pentose assay appears to be a simple and convenient colorimetric assay for detecting any pentose-rich glycoprotein in plant extracts. The utility of the pentose assay appears to have a significant potential in the detection of hydroxyproline-rich glycoproteins (HRGPs), which are generally O-arabinosylated.     

Environmental drivers of paralytic shellfish toxin producing Alexandrium catenella blooms in a fjord system of northern Southeast Alaska

Paralytic shellfish poisoning (PSP) is a persistent problem that threatens human health and the availability of shellfish resources in Alaska. Regular outbreaks of marine dinoflagellates in the genus Alexandrium produce paralytic shellfish toxins (PSTs) that make shellfish consumption unsafe, and impose economic hardships on Alaska’s shellfish industry. Phytoplankton and environmental monitoring spanning 2008–2016, and a pilot benthic cyst survey in 2016, were focused in the Juneau region of Southeast Alaska to investigate Alexandrium catenella distributions and conditions favorable to bloom development. Overwintering Alexandrium cysts were found in near-shore sediments throughout the study region. Alexandrium catenella cells were present in the water column across a range of sea surface temperatures (7–15 °C) and surface salinities (S = 4–30); however, an optimal temperature/salinity window (10–13 °C, 18–23) supported highest cell concentrations. Measurable levels of PSTs were associated with lower concentrations (100 cells L⁻¹) of A. catenella, indicating high cell densities may not be required for shellfish toxicity to occur. Several interacting local factors were identified to support A. catenella blooms: 1) sea surface temperatures ≥7 °C; 2) increasing air temperature; 3) low to moderate freshwater discharge; and 4) several consecutive days of dry and calm weather. In combination, these bloom favorable conditions coincide with toxic bloom events during May and June in northern Southeast Alaska. These findings highlight how integrated environmental and phytoplankton monitoring can be used to enhance early warning capacity of toxic bloom events, providing more informed guidance to shellfish harvesters and resource managers in Alaska.     

Preparation and Analysis of an Expressed Sequence Tag Library from the Toxic Dinoflagellate <Emphasis Type="Italic">Alexandrium catenella</Emphasis>

Dinoflagellates of the genus Alexandrium are photosynthetic microalgae that have an extreme importance due to the impact of some toxic species on shellfish aquaculture industry. Alexandrium catenella is the species responsible for the production of paralytic shellfish poisoning in Chile and other geographical areas. We have constructed a cDNA library from midexponential cells of A. catenella grown in culture free of associated bacteria and sequenced 10,850 expressed sequence tags (ESTs) that were assembled into 1,021 contigs and 5,475 singletons for a total of 6,496 unigenes. Approximately 41.6% of the unigenes showed similarity to genes with predicted function. A significant number of unigenes showed similarity with genes from other dinoflagellates, plants, and other protists. Among the identified genes, the most expressed correspond to those coding for proteins of luminescence, carbohydrate metabolism, and photosynthesis. The sequences of 9,847 ESTs have been deposited in Gene Bank (accession numbers EX 454357–464203).     

Characterization of a Cloned pR72H Probe for Vibrio parahaemolyticus Detection and Development of a Nonisotopic Colony Hybridization Assay

Vibrio parahaemolyticus is a halophilic bacterium often found in shellfish and is an important causative agent of food poisoning in Taiwan. A rapid and efficient detection method is required to identify this foodborne pathogen. A 0.76-Kb HindIII DNA fragment was cloned from the chromosomal DNA of V. parahaemolyticus strain no. 93, designated as pR72H fragment, was used as a polynucleotide probe. It was labeled with digoxigenin-11-dUTP (DIG) by the random primer-labeling method. The sensitivity and specificity of the digoxigenin-labeled 0.76-Kb DNA probe was determined by colony hybridization assay. Under stringent hybridization conditions, 122 of 124 isolates of V. parahaemolyticus showed positive hybridization reaction with DIG-0.76-Kb DNA probe; the negative strains were attributed to slow growth. The DIG-0.76-Kb probe did not hybridize with 86 isolates of other vibrios and a number of other enterics as well as nonenteric microorganisms. The sensitivity and specificity of this DIG probe are 98% and 100%, respectively. This nonisotopic colony hybridization assay can be very useful for routine monitoring of V. parahaemolyticus in the food industry, environmental analysis and clinical laboratories.