Multiplex PCR for the detection of toxigenic cyanobacteria in dietary supplements produced for human consumption

The production of food supplements containing cyanobacteria is a growing worldwide industry. While there have been several reports of health benefits that can be gained from the consumption of these supplements, there have also been a growing number of studies showing the presence of toxins some of which (for example microcystins) are known to affect human health. In this paper, we report a multiplex polymerase chain reaction (PCR) technique that can be used to identify microcystin contamination in dietary supplements produced for human consumption. This method involves a PCR reaction containing three primer pairs, the first of which is used to amplify a 220-bp fragment of 16s rDNA specific to Microcystis, the most common microcystin-producing cyanobacterium. The second primer pair is used to amplify a 300-bp fragment of the mcyA gene, linked to microcystin biosynthesis in Anabaena, Microcystis, and Planktothrix. A third primer pair, used as a positive control, results in the amplification of a 650-bp fragment from the phycocyanin operon common to all cyanobacteria. This technique was found to be useful for detecting the presence of toxigenic Microcystis in all dietary supplements produced from the nontoxic cyanobacterium Aphanizomenon flos-aquae.     

PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera

Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.Abbreviations RFLP Restriction fragment length polymorphism - MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight spectrometry - HPLC High performance liquid chromatography     

Detection of potential microcystin-producing cyanobacteria in Brazilian reservoirs with a mcyB molecular marker

One of the most serious problems related to water eutrophication is the occurrence of increasingly frequent blooms of toxic cyanobacteria in freshwater ecosystems. Microcystin (MCYST) molecular markers may be used for the detection of toxic cyanobacteria, both cultivated strains and environmental samples, independently of their taxonomic category and production of the toxin at the moment of analysis. Sixty Microcystis spp. strains from 15 water reservoirs of south, southeastern and northeastern Brazil were analyzed by polymerase chain reaction (PCR) with oligonucleotide primers for mcyB gene of the operon that encodes a microcystin synthetase. It was found out that the presence of a unique amplified product of approximately 780 bp in 18 strains, indicated the presence of the microcystin-producing genotype. There was correspondence between the presence of the mcyB gene and microcystin determined by ELISA. Eight reservoirs contained toxic strains, two of these reservoirs being used mainly for public water supply. The coexistence of a mixture of toxic and non-toxic genotypes in populations of several reservoirs was found. Thus, it is evident that Microcystis populations present in blooms compose a mosaic, with genetically different individuals within the same population, each one, possibly, with its own tolerance to environmental factors and with distinct toxicity potential.     

Quantification of toxigenic Microcystis spp. in freshwaters by quantitative real-time PCR based on the microcystin synthetase A gene

A method to estimate the abundance of toxigenic Microcystis in environmental samples by using quantitative real-time PCR was developed and optimized. The basis of this method is the amplification of a highly conserved region of the mcyA gene within the microcystin synthetase gene cluster. Using this method, the average copy number of mcyA gene per cell in toxigenic Microcystis strains was estimated. The molecular markers and method developed in this study can be used to monitor toxigenic strains of Microcystis in Korean freshwaters, in which harmful cyanobacterial blooms are routinely found.     

Lake Erie Microcystis: Relationship between microcystin production, dynamics of genotypes and environmental parameters in a large lake

Cyanobacteria of genus Microcystis sp. have been commonly found in Lake Erie waters during recent summer seasons. In an effort to elucidate relationships between microcystin production, genotypic composition of Microcystis community and environmental parameters in a large lake ecosystem, we collected DNA samples and environmental data during a three-year (2003–2005) survey within Lake Erie and used the data to perform a series of correlation analyses. Cyanobacteria and Microcystis genotypes were quantified using quantitative real-time PCR (qPCR). Our data show that Microcystis in Lake Erie forms up to 42% of all cyanobacteria, and that Microcystis exists as a mixed population of potentially toxic and (primarily) non-toxic genotypes. In the entire lake, the total abundance of Microcystis as well as the abundance of microcystin-producing Microcystis is strongly correlated with the abundance of cyanobacteria suggesting that Microcystis is a significant component of the cyanobacterial community in Lake Erie during summer seasons. The proportion of total Microcystis of all cyanobacteria was strongly linked to the microcystin concentrations, while the percentage of microcystin-producing genotypes within Microcystis population showed no correlation with microcystin concentrations. Correlation analysis indicated that increasing total phosphorus concentrations correlate strongly with increasing microcystin concentrations as well as with the total abundance of Microcystis and microcystin-producing Microcystis.     

Development and application of a multiplex qPCR technique to detect multiple microcystin-producing cyanobacterial genera in a Canadian freshwater lake

The emergence and persistence of complex blooms comprising multiple toxigenic cyanobacteria genera pose significant challenges for water quality management worldwide. The co-occurrence of morphologically indistinguishable toxic and non-toxic strains makes monitoring and control of these noxious organisms particularly challenging. Conventional monitoring approaches are not only incapable of discriminating toxic from non-toxic strains but also have proven to be less sensitive and specific. In this study, a multiplex quantitative real-time polymerase chain reaction (qPCR) approach was developed and tested for its sensitivity and specificity at detecting, differentiating and estimating potentially toxic Anabaena, Microcystis and Planktothrix genotype compositions in environmental samples. The oligonucleotide primers and probes utilized were designed to target portions of the microcystin synthetase (mcy) E gene that encode synthesis of the unique 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA) moiety of microcystins in the three target genera. Laboratory evaluation showed the developed assay to be highly sensitive and specific at detecting and quantifying targeted genera. Indeed, the assay standards for the Anabaena, Microcystis and Planktothrix reactions attained efficiencies above 90 %, with coefficients of determination consistently above 0.99. Analysis of water samples from Missisquoi Bay, Quebec, Canada, resulted in successful detection and quantification of target toxigenic cyanobacteria even when cell numbers were below the detection limit for the conventional microscopy methods. Furthermore, toxigenic Microcystis spp. were found to be the main putative microcystin-producing cyanobacteria in the study lake. The qPCR technique developed in this study therefore offers simultaneous detection, differentiation and quantification of multiple toxigenic cyanobacteria that otherwise cannot be accomplished by current monitoring approaches.     

The diversity and distribution of toxigenic Microcystis spp. in present day and archived pelagic and sediment samples from Lake Erie

The reoccurrence of significant cyanobacterial blooms in Lake Erie during the last 13 years has raised questions concerning the long-term persistence of microcystin-producing cyanobacteria and the presence of natural sediment reservoirs for potentially toxic cyanobacteria in this large lake system. To address these questions, we analyzed phytoplankton and sediment samples which were collected and preserved in the 1970s as well as samples collected in 2004 from locations within Lake Erie. The identification of microcystin-producing cyanobacteria in Lake Erie was examined via PCR amplification of the mcyA gene fragment. Based on the high % sequence similarity, the mcyA sequences from all 1970s phytoplankton and sediment samples were determined to belong to Microcystis spp., in spite of reports suggesting that Lake Erie was dominated by filamentous cyanobacteria in the 1970s. In sediment samples from 2004, signature genes for Microcystis were distributed and preserved not only in the surface sediments but also up to 10–12 cm in depth. Based on cell quantities determined by a quantitative polymerase chain reaction (qPCR) method, 0.18% of eubacteria in the sediments were Microcystis cells, of which 4.8% were potential microcystin producers. In combination with experiments showing that Microcystis cells can be cultured from Lake Erie surface sediments, this paper demonstrates the potential for these sediments to act as a reservoir for pelagic Microcystis populations and that the composition of the population of microcystin-producing cyanobacteria in Lake Erie has not changed remarkably since the 1970s.     

Identification and enumeration of <Emphasis Type="Italic">Microcystis</Emphasis> using a sandwich hybridization assay

Based on sequence analyses of phycocyanin intergenic spacers (PC-IGS) from Microcystis, Anabaena, Aphanizomenon, and Planktothrix (Oscillatoria) strains, a genus-specific probe pair TF/TR was designed, and a sandwich hybridization assay was established to quantitatively detect Microcystis. Through BLAST and cyanobacterial culture tests, TF/TR was demonstrated to be specific for Microcystis. A calibration curve for the sandwich hybridization assay was established, and the lowest detected concentration was 100 cell/ml. Laboratory and field samples were analyzed with both sandwich hybridization assay and microscopy. The biotic and abiotic components of the samples were of little disturbance to the sandwich hybridization assay. The results showed no distinct difference between the two methods. In this study, a sandwich hybridization assay was established to detect Microcystis, providing an alternative to traditional microscopic, morphology-based methods.     

Typing of toxinogenic Microcystis from environmental samples by multiplex PCR

Microcystin (MC)-producing Microcystis strains from environmental samples were assessed by the simultaneous amplification of up to five DNA sequences, corresponding to specific regions of six mcy genes (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), codifying for key motifs of the non-ribosomal peptide synthetase and polyketide synthase of the microcystin synthetase complex. Six primer pairs with the same melting temperature, one of them of new design, were used. A crucial point for the good performance of the new multiplex PCR test was the concentration of each primer pair. In the test, cell suspensions from laboratory cultures, field colonies and blooms were directly used as DNA source. The results of the multiplex PCR were consistent with the toxinogenic character of the samples, as checked by high performance liquid chromatography and/or matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. As a whole, the newly developed test could be used for a reliable, rapid and low-cost screening of potential MC-producing Microcystis in field samples, even scattered colonies.     

Detection of Microcystis in Lake Sediment using Molecular Genetic Techniques

Summary Microcystis, which are toxic microcystin-producing cyanobacteria, normally bloom in summer and drop in numbers during the winter season in Senba Lake, Japan. Recently, this lake has been treated by ultrasonic radiation and jet circulation which were integrated with flushing with river water. This treatment was most likely sufficient for the destruction of cyanobacterial gas vacuoles. In order to confirm whether Microcystis viridis was still present, a molecular genetic monitoring technique on the basis of DNA direct extraction from the sediment was applied. Three primer sets were used for polymerase chain reaction (PCR) based on rRNA intergenic spacer analysis (RISA), the DNA dependent RNA polymerase (rpoC1) and a Microcystis sp.-specific rpoC1 fragment. The results from each primer were demonstrated on the basis of single strand conformation polymorphisms (SSCP). Using the RISA primer showed different results from the rpoC1 and Microcystis sp.-specific rpoC1 fragment; meanwhile, the rpoC1 Microcystis sp.-specific fragment was more specific than the RISA primer. Therefore, the Microcystis sp.-specific rpoC1 fragment was further analysed by denaturing gradient gel electrophoresis (DGGE). The DNA pattern representing M. viridis could not be detected in any of the sediment samples. However, the results were confirmed with another technique, terminal restriction fragment length polymorphisms (T-RFLP). Although T-RFLP patterns of 16S rDNA in sediment at 91 bp and 477 bp lengths were matched with the T-RFLP of M. viridis (HhaI and MspI endonuclease digestion, respectively), the T-RFLP pattern of 75 bp length was not matched with M. viridis (both of HhaI and MspI endonuclease digestion) which were the major T-RFLP pattern of M. viridis. Therefore, the results most likely indicated that M. viridis seems to have disappeared because of the addition of the ultrasonic radiation and jet circulation to the flushing treatment.     

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.     

LOCALIZATION OF MICROCYSTIN SYNTHETASE GENES IN COLONIES OF THE CYANOBACTERIUM MICROCYSTIS USING FLUORESCENCE IN SITU HYBRIDIZATION1

To better understand the production of microcystins (MCs) in Microcystis colonies, fluorescence in situ hybridization (FISH) methods were developed to detect DNA involved in the synthesis of these cyanobacterial hepatotoxins. Using colonies of Microcystis aeruginosa (Kütz.) Kütz. isolated from environmental blooms of cyanobacteria and from a colony‐forming, MC‐producing laboratory strain of Microcystis, amplified PCR products were observed, coincident with positive controls. The total MC content of individual colonies of Microcystis, determined by ELISA, showed a positive correlation with colony cross‐sectional area. FISH analysis of Microcystis colonies gave high fluorescence in comparison to negative controls, indicating the presence of MC synthetase DNA (mcyA) in situ. FISH analysis for MC synthetase genes has the potential to be developed into an effective early warning tool for drinking and recreational water management.     

Semi-nested polymerase chain reaction for detection of toxigenic <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from environmental water samples

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10³ CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10⁴ CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.     

Incidence of microcystin‐producing cyanobacteria in Lake Tana,the largest waterbody in Ethiopia

The occurrence of toxic cyanobacterial blooms is a serious problem for fast‐developing countries in Africa, such as Ethiopia, that are struggling with significant degradation of the natural environment and limited access to water of good quality. Research undertaken on Lake Tana in Ethiopia between 2009 and 2011 was intended to assess the seasonal threat from cyanobacteria and to select methods for tracking of this threat in the future. The cyanobacterial genus Microcystis was found to be present throughout the monitoring period, and M. aeruginosa was determined as the dominant species. Moreover, in all samples, toxigenic cyanobacteria with the potential to produce microcystins were detected. High levels of microcystins, ranging from 0.58 to 2.65 μg L^?1, were detected each November, which indicates that in the postrainy season, water usage should be limited. The correlation between concentrations of chlorophyll‐a and microcystins suggested that chlorophyll‐a could be used as an indicator of the potential presence of cyanobacterial‐derived hepatotoxins in Lake Tana in the future. Furthermore, for quick quantitative confirmation of the presence of microcystins, a simple and rapid ELISA test was recommended.     

Development of tolerance against toxic cyanobacteria in Daphnia

We tested whether previous exposure to a toxic strain of cyanobacteria (Microcystis) affects survival, growth, and reproduction of a common herbivore, Daphnia magna. Samples from three natural populations of D. magna were each divided into two parts; one part was fed a mixture of toxic Microcystis and the non-toxic green alga Scenedesmus whereas the other part was fed only Scenedesmus. After four weeks, we compared the ability of these two populations to withstand the toxic Microcystis by assessing survivorship, growth, and reproduction. We found that the ability of D. magna to cope successfully with toxic Microcystis is improved if the animals have experienced previous exposure to toxic Microcystis. This suggests that the toxin may less affect the D. magna populations that are repeatedly exposed to toxic cyanobacteria in their natural habitat than populations lacking prior exposure. Since the ability to tolerate toxins is manifested in both improved survival and larger size of the animals, it may have considerable impact on zooplankton community composition in fresh-waters.     

Toxic Microcystis is Widespread in Lake Erie: PCR Detection of Toxin Genes and Molecular Characterization of Associated Cyanobacterial Communities

During the past decade, algae blooms, which include the toxic cyanobacterium Microcystis, have reoccurred in the Laurentian Great Lakes, most commonly in the western basin of Lake Erie. Whereas the western basin is the most impacted by toxic Microcystis in Lake Erie, there has historically been little effort focused on identifying the spatial distribution of Microcystis throughout this lake. To address this lack of knowledge, we have employed a polymerase-chain-reaction-based detection of genes required for synthesis of the toxin microcystin (mcyD and mcyB), as well as 16S rDNA fragments specific to either all Microcystis or all cyanobacteria. Using a multiplex approach, we tested 21 samples from 13 field stations and found that toxigenic Microcystis were present in the western and eastern basins in the summers of 1999, 2000, and 2002 and the central basin in 1999 and 2002. This is the most extensive distribution of Microcystis reported in Lake Erie. Clone libraries (16S rDNA) of these cyanobacterial communities were generated from 7 of the 13 field stations (representing all three basins) to partially characterize this microbial community. These libraries were shown to be dominated by sequences assigned to the Synechococcus and Cyanobium phylogenetic cluster, indicating the importance of picoplankton in this large lake system.     

Rapid detection and identification of the free-living nitrogen fixing genus <Emphasis Type="Italic">Azospirillum</Emphasis> by 16S rRNA-gene-targeted genus-specific primers

The modern agricultural practice utilizing plant growth promoting rhizobacteria (PGPR) has brought great benefits in the promotion of crop growth. Among PGPR, Azospirillum is considered as an important genus which is not only closely-associated with plants but also shows potential in the degradation of organic contaminants. However, lack of media for selective isolation or techniques for specific detection or identification limit the exploration of these rhizobacteria. This motivated us to design a genus-specific oligonucleotide primer pair which could assist in rapid detection of species of the genus Azospirillum by means of PCR-specific amplification. The sensitivity and specificity of the newly designed primer pair Azo494-F/Azo756-R were tested against 12 Azospirillum type strains and other closely-related genera. The Azospirillum-specific 16S rRNA gene fragment (263 bp) was successfully amplified for all the reference Azospirillum species with the designed primer pair. No amplification was noted for closely-related species from other genera. The genus specificity was validated with 18 strains including environmental isolates. Interestingly, two strains assigned earlier as Azospirillum amazonense (DSM 2787^T) and Azospirillum irakense (DSM 11586^T) failed to produce an Azospirillum-specific fragment with this primer pair. Further phylogenetic analysis of these two isolates based on 16S rRNA gene sequences shows that these two strains might belong to other genera rather than Azospirillum. Preliminary screening of isolates and soil samples with the Azospirillum-specific primers was successful in terms of the rapid detection of Azospirillum isolates. By using real-time PCR analysis the minimum limit of Azospirillum detection was 10² CFU g⁻¹ in the seeded soil sample. The newly designed primers can be used to study the diversity of Azospirillum in ecosystems and aid in the exploration of novel species.     

High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3′ end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.     

Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products

A multiplex polymerase chain reaction (MPCR)-based assay was developed for the simultaneous detection of Vibrios using the genus-specific RNA polymerase subunit A (rpoA) gene and specific detection of toxin-producing Vibrio cholerae strains using two sets of primer based on cholera toxin subunit A (ctxA) and repeat in toxin subunit A (RtxA)-producing genes. The MPCR method developed is applicable to both the simultaneous and the two-step detection of genus Vibrio total and toxigenic V. cholerae species. This assay was specific as no amplification occurred with the other bacterial pathogens tested. The sensitivity of the assay was tested by artificially spiking the shrimp homogenate with the toxigenic strain of V. cholerae (NICED 16582) in different dilutions. The developed MPCR assay could detect three cells of V. cholerae in 12 h pre-enrichment in APW. The proposed method is rapid, sensitive, and specific for the detection of Vibrio genus as well as toxin-producing V. cholerae strains in environmental samples.