Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

• compositions for detecting dinoflagellates by amplification of the sxtG gene are provided.
• Saxitoxin commonly known as paralytic shellfish poison (PSP), and its derivatives, are environmental neurotoxins that can cause severe symptoms upon consumption of vector species (see, e.g., Deeds JR, et al. (2008), “Non- traditional vectors for paralytic shellfish poisoning", Marine Drugs 6- 308-348; Wiese M, et al. (2010) “Neurotoxic Alkaloids : Saxitoxin and Its Analogs", Marine Drugs 8' ⁇ 2185-2211). These toxic compounds accumulate in shellfish and in other marine organisms that feed on dinoflagellates.
• dinoflagellates Conventional detection of dinoflagellates comprises regular collection of water samples and manual analysis to detect the presence of potentially toxic dinoflagellate species. If dinoflagellates are present in sufficient numbers, the aquaculture area affected (e.g., a mussel bed) is closed. In addition, chemical methods (e.g., HPLC and LCMS) are often used to measure the amount of saxitoxin in affected tissues (e.g., mussel tissue). Sometimes a mouse bioassay is used.
• sxtG gene is an addition to the sxtA gene previously identified.
• a double assay based on detecting both sxtA and sxtG provides increased accuracy in
• a dinoflagellate saxitoxin G polynucleotide or a polypeptide encoded by said polynucleotide wherein the presence of said polynucleotide or polypeptide indicates the presence of a saxitoxin-producing dinoflagellate in the sample and wherein the absence of said polynucleotide or polypeptide indicates the absence of a saxitoxin-producing dinoflagellate in the sample.
• the polynucleotide comprises a saxitoxin G nucleotide sequence selected from those set forth in any one of SEQ ID NOs : 12-13, or a fragment or variant of any one of those sequences.
• the method further comprises analyzing the sample for the presence or absence of one or more of a dinoflagellate saxitoxin A polynucleotide or a polypeptide encoded by said polynucleotide.
• some embodiments provide a method wherein said analyzing comprises amplification of polynucleotides from the sample by polymerase chain reaction.
• the polymerase chain reaction utilises one or more primers comprising a sequence set forth in SEQ ID NOs : 1-9, or a fragment or variant of any of these sequences.
• the polypeptide comprises a saxitoxin G amino acid sequence, or a fragment or variant thereof.
• the saxitoxin producing dinoflagellate is from the genus Alexandrium, Pyrodinium, or Gymnodinium.
• the saxitoxin-producing dinoflagellate is selected from the group consisting of A catenella, A. fundyense, A. lusitanicum, A. minutum, A.
• Embodiments of the technology also comprise kits for the detection of a saxitoxin-producing dinoflagellate in a sample or for determining the absence of a saxitoxin-producing dinoflagellate in a sample, the kits comprising at least one agent for detecting the presence of a dinoflagellate saxitoxin G polynucleotide or a polypeptide encoded by said polynucleotide.
• the agent binds specifically to a polynucleotide comprising a saxitoxin G nucleotide sequence selected from those set forth in any one of SEQ ID NOs : 12-13, or a fragment or variant of any one of those sequences.
• the agent binds specifically to a polynucleotide encoding a saxitoxin G peptide sequence or a fragment thereof. Additional embodiments further comprise at least one agent for detecting the presence of a dinoflagellate saxitoxin A polynucleotide or a polypeptide encoded by said polynucleotide.
• the agent is a primer, a probe, or an antibody. Specific embodiments provide an agent that is a primer comprising a sequence set forth in SEQ ID NOs : 1-9, or a fragment or variant of any of these sequences.
• the agent binds specifically to a saxitoxin G amino acid sequence, or a fragment or variant thereof.
• the technology encompasses an isolated polynucleotide comprising a sequence set forth in SEQ ID NO : 12 or 13, or a variant or fragment thereof and an isolated polynucleotide comprising the sequence set forth in any one of SEQ ID NOS : 1-9, or a variant or fragment of any one of those sequences.
• embodiments include an isolated polypeptide encoded by these polynucleotides.
• Figure 1 is a sequence comprising sxtGim A. fundyense.
• Figure 2 is a sequence comprising sxtGirom A. minutum.
• compositions for detecting dinoflagellates by amplification of the sxtG gene are provided.
• saxitoxin encompasses pure saxitoxin and analogs of thereof, non-limiting examples of which include neosaxitoxin
• neoSTX gonyautoxins
• dcSTX decarbamoylsaxitoxin
• STX refers to a saxitoxin
• SXT refers to a gene product (e.g., a polypeptide) of an sxt gene such as sxtGaxidJor sxtA.
• a polynucleotide "fragment" as contemplated herein is a polynucleotide molecule that is a constituent of a polynucleotide of the technology or variant thereof. Fragments of a polynucleotide do not necessarily need to encode polypeptides which retain biological activity although this is not excluded from being the case. In certain embodiments the fragment may be useful as a hybridization probe or PCR primer. The fragment may be derived by cleaving a polynucleotide of the technology or alternatively may be synthesized by some other means, for example by chemical synthesis.
• a polynucleotide fragment as contemplated herein may be less than about 5000 nucleotides in length, less than about 4500 nucleotides in length, or less than about 4000, 3500, 3000, 2500, 2000, 1500, 1000, 40 750, 500, 400, 300, 250, 200, 150, 100, 75, 50, 25 or 15 nucleotides in length.
• a polynucleotide fragment as contemplated herein may be more than about 15 nucleotides in length, more than about 25 nucleotides in length, or more than about 50, 75, 100, 150, 200, 250, 300, 400, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500 or 4000 nucleotides in length. Additionally or alternatively, a polynucleotide fragment as
• Polynucleotide fragments of the technology comprise fragments of the sxtG gene.
• a polypeptide "fragment” as contemplated herein is a polypeptide molecule is a constituent of a polypeptide of the technology or variant thereof. Typically the fragment possesses qualitative biological activity in common with the polypeptide of which it is a constituent though this is not necessarily required.
• a polypeptide fragment as contemplated herein may be less than about 1500 amino acid residues in length, less than about 1400 amino acid residues in length, or less than about 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100, 75, 50, 25 or 15 amino acid residues in length.
• a polypeptide fragment as contemplated herein may be more than about 15 amino acid residues in length, more than about 25 amino acid residues in length, or more than about 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acid residues in length.
• a polypeptide fragment as contemplated herein may be between about 15 and about 25 amino acid residues in length, between about 15 and about 50 amino acid residues in length, or between about 15 and 75, 15 and 100, 15 and 150, 25 and 50, 25 and 100, 50 and 100, 50 and 150, 100 and 200, 100 and 250, 100 and 300, 100 and 500, 500 and 750, 500 and 1000, or 1000 and 1300 amino acid residues in length.
• Polypeptide fragments of the technology comprise fragments of the sxtG protein or the saxitoxin protein.
• dinoflagellate saxitoxin polynucleotide and polypeptide sequences are disclosed herein.
• Polynucleotides of the technology may be
• sequences are saxitoxin G gene (sxtG) polynucleotide sequences or saxitoxin G polypeptide (SXTG) sequences.
• sxtG saxitoxin G gene
• SXTG saxitoxin G polypeptide
• the polynucleotide and polypeptide sequences are from saxitoxin producing dinoflagellates.
• the polynucleotide and polypeptide sequences may be from dinoflagellates of the order Gonyaulacales or Gymnodiniales.
• the dinoflagellates are of the genus Alexandrium (formerly Gonyaulax) , Pyrodinium, or Gymnodinium. sxtG
• sxtA the starting gene of saxitoxin synthesis
• dinoflagellates e.g., Gymnodinium catenatum and multiple Alexandrium species
• sxtG the second core gene in the STX synthesis pathway.
• sxtG was predicted to encode an amidinotransferase domain.
• the product of sxtA is the substrate for the amidinotransferase sxtG.
• SxtG is proposed to incorporate an amidino group from a second arginine molecule into the STX intermediate.
• the sxt G transcript possessed a eukaryotic polyA-tail at the 3' end and the
• sxtG amidinotransferase was present and transcribed in all tested
• Alexandrium species including those where sxtA and STX synthesis were undetectable (Stuken A, et al. (2011) “Discovery of Nuclear- Encoded Genes for the Neurotoxin Saxitoxin in Dinoflagellates” PlosOne 6' ⁇ e20096; Orr RJS, et al. (2011) “Improved phylogenetic resolution of toxic and non-toxic Alexandrium strains using a concatenated rDNA approach", Harmful Algae 10: 676-688).
• sxtG was not detected. Twenty-two species from five orders are seemingly devoid of these genes (Fig. 2). Further, only significant hits to Alexandrium were produced upon blasting multiple databases within
• Genbank with the sxtG query While sxtG is not exclusive to toxic species, it is present in all Alexandrium species and it is absent from non-PSP dinoflagellate genera.
• variants of polynucleotides of the technology and polypeptides of the technology, and fragments thereof, are also provided herein.
• a “variant” as contemplated herein refers to a substantially similar sequence. In general, two sequences are “substantially similar” if the two sequences have a specified percentage of amino acid residues or nucleotides that are the same (percentage of "sequence identity"), over a specified region, or, when not specified, over the entire sequence.
• a "variant" of a polynucleotide and polypeptide sequence disclosed herein may share at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83% 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity with the reference sequence.
• polypeptide sequence variants possess qualitative biological activity in common.
• Polynucleotide sequence variants generally encode polypeptides which generally possess qualitative biological activity in common.
• variants are homologues of polynucleotides of the technology and polypeptides of the technology.
• polynucleotide homologue is typically from a different dinoflagellate species but sharing substantially the same biological function or activity as the
• a polypeptide homologue is typically from a different dinoflagellate species but sharing substantially the same biological function or activity as the corresponding polypeptide disclosed herein.
• variant also includes analogues of the polypeptides of the technology.
• a polypeptide "analogue” is a polypeptide which is a derivative of a polypeptide of the technology, which derivative comprises addition, deletion, substitution of one or more amino acids, such that the polypeptide retains substantially the same function.
• conservative amino acid substitution refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain (primary sequence of a protein).
• polynucleotides of the technology and polypeptides of the technology are “isolated”. It will be understood that the term “isolated” in this context means that the polynucleotide or polypeptide has been removed from or is not associated with some or all of the other components with which it would be found in its natural state.
• an “isolated” polynucleotide may be removed from other polynucleotides of a larger polynucleotide sequence, or may be removed from natural components such as unrelated polynucleotides.
• an "isolated" polypeptide may be removed from other polypeptides of a larger polypeptide sequence, or may be removed from natural components such as unrelated polypeptides.
• an "isolated" polynucleotide of polypeptide also includes a polynucleotide or polypeptide which has not been taken from nature but rather has been prepared de novo, such as chemically synthesised and/or prepared by recombinant methods.
• an isolated polypeptide of the technology may be included as a component part of a longer polypeptide or fusion protein.
• polynucleotides of the technology may be cloned into a vector.
• the vector may comprise, for example, a DNA, RNA or
• the vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences, their introduction into cells and the expression of the introduced sequences.
• the vector is an expression vector and may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyadenylation signals and transcription termination sequences.
• the technology also contemplates host cells transformed by such vectors.
• the polynucleotides of the technology may be cloned into a vector which is transformed into a bacterial host cell, for example E. coli.
• Polynucleotides of the technology include derivatives and fragments thereof for use as primers and probes.
• the derivatives and fragments may be in the form of oligonucleotides.
• Oligonucleotides are short stretches of nucleotide residues suitable for use in nucleic acid amplification reactions such as PCR, typically being at least about 5 nucleotides to about 80 nucleotides in length, more typically about 10 nucleotides in length to about 50 nucleotides in length, and even more typically about 15 nucleotides in length to about 30 nucleotides in length.
• Probes are nucleotide sequences of variable length, for example between about 10 nucleotides and several thousand nucleotides, for use in detection of homologous sequences, typically by hybridization.
• Hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other
• nucleotide probes and/or primers are known in the art, and described in standard texts such as Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York; and publications such as Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Innis et al.
• Polynucleotide primers and probes may be prepared, for example, by chemical synthesis techniques such as the phosphodiester and phosphotriester methods (see for example Narang et al. (1979) Meth. Enzymol. 68 : 90; Brown et al. (1979) Meth. Enzymol. 68:109; and U.S. Patent No.
• Polynucleotides of the technology may be labeled by incorporation of a marker to facilitate their detection.
• a marker to facilitate their detection.
• Techniques for labelling and detecting nucleic acids are described, for example, in standard texts such as Ausubel et al. (Eds) Current Protocols in Molecular Biology (2007), John Wiley and Sons, Inc..
• suitable markers include fluorescent molecules (e.g.
• acetylaminofluorene 5-bromodeoxyuridine, digoxigenin, and fluorescein
• radioactive isotopes e.g. 32 P, 35 S, 3 H, 33 P.
• Detection of the marker may be achieved, for example, by chemical, photochemical, immunochemical,
• the probes and primers may be used, for example, to detect or isolate dinoflagellates in a sample of interest.
• the probes and primers may be used to detect STX-producing dinoflagellates in a sample of interest.
• the probes or primers may be used to isolate corresponding sequences in other organisms including, for example, other dinoflagellate species.
• Methods such as the polymerase chain reaction (PCR), hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences that are selected based on their sequence identity to the entire sequences set forth herein or to fragments thereof are encompassed by the embodiments.
• sequences include sequences that are orthologs of the disclosed sequences.
• orthologs refers to genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein.
• hybridization techniques all or part of a known nucleotide sequence is used to generate a probe that selectively hybridizes to other corresponding nucleic acid sequences present in a given sample.
• the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable marker.
• probes for hybridization can be made by labelling synthetic
• oligonucleotides based on the sequences of the technology.
• the level of homology (sequence identity) between probe and the target sequence will largely be determined by the stringency of hybridization conditions.
• the nucleotide sequence used as a probe may hybridize to a homologue or other variant of a polynucleotide disclosed herein under conditions of low stringency, medium stringency or high stringency.
• oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest.
• Methods for designing PCR primers and PCR cloning are generally known in the art.
• Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector- specific primers, partially-mismatched primers, and the like.
• the skilled addressee will recognise that the primers described herein for use in PCR or RT-PCR may also be used as probes for the detection of dinoflagellate sxt gene sequences.
• antibodies which are capable of binding specifically to polypeptides of the technology.
• the antibodies may be used to qualitatively or quantitatively detect and analyse one or more STX polypeptides (e.g., toxin) in a given sample and/or one or more SXT polypeptides (e.g., a product of the sxtG gene and/or a product of the sxtA gene) in a sample.
• STX polypeptides e.g., toxin
• SXT polypeptides e.g., a product of the sxtG gene and/or a product of the sxtA gene
• the antibody may bind to the polypeptide or fragment thereof with a binding constant in the range of at least about 10" 4 M to about 10 ⁇ 10 M.
• the binding constant is at least about 10" 5 M, or at least about 10 ⁇ 6 M. More preferably the binding constant is at least about 10" 7 M, at least about 10 ⁇ 8 M, or at least about 10 ⁇ 9 M or more.
• reference to an antibody specific to a particular polypeptide includes, e.g., an antibody that is specific to a fragment of the polypeptide.
• Antibodies of the technology may exist in a variety of forms including, for example, as a whole antibody, or as an antibody fragment, or other
• the antibody may exist as an antibody fragment having functional antigen-binding domains, that is, heavy and light chain variable domains.
• the antibody fragment may exist in a form selected from the group consisting of, but not limited to : Fv, F a b, F(ab)2, scFv (single chain Fv), dAb (single domain antibody), chimeric antibodies, bi- specific antibodies, diabodies and triabodies.
• An antibody "fragment” may be produced by modification of a whole antibody or by synthesis of the desired antibody fragment.
• Methods of generating antibodies, including antibody fragments are known in the art and include, for example, synthesis by recombinant DNA technology. The skilled addressee will be aware of methods of synthesising antibodies, such as those described in, for example, US Patent No. 5296348 and standard texts such as Ausubel et al. (Eds) Current Protocols in Molecular Biology (2007), John Wiley and Sons, Inc.
• antibodies are prepared from discrete regions or fragments of the SxtG or SXT polypeptide of interest.
• An antigenic portion of a polypeptide of interest may be of any appropriate length, such as from about 5 to about 15 amino acids.
• an antigenic portion contains at least about 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid residues.
• Antibodies that specifically bind to a polypeptide of the technology can be prepared, for example, using purified SXT polypeptides of the technology, or purified sxt polynucleotide sequences of the technology that encode SXT polypeptides of the technology using any suitable methods known in the art.
• a monoclonal antibody typically containing F a b portions, may be prepared using hybridoma technology described in Harlow and Lane (Eds) Antibodies - A Laboratory Manual, (1988), Cold Spring Harbor Laboratory, N.Y; Coligan, Current Protocols in Immunology (1991); Goding, Monoclonal
• Such techniques include, but are not limited to, antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, for example, Huse et al. (1989) Science 246: 1275-1281; Ward et al. (1989) Nature 341: 544-546).
• antibodies of the technology include humanised antibodies, chimeric antibodies and fully human antibodies.
• An antibody of the technology may be a bi-specific antibody, having binding specificity to more than one antigen or epitope.
• the antibody may have specificity for one or more STX polypeptides or fragments thereof, and additionally have binding specificity for another antigen.
• Methods for the preparation of humanised antibodies, chimeric antibodies, fully human antibodies, and bispecific antibodies are known in the art and include, for example, those described in United States Patent No. 6995243.
• a sample potentially comprising an STX polypeptide of the technology can be contacted with an antibody that specifically binds the STX polypeptide or fragment thereof.
• the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample.
• solid supports include, for example, microtitre plates, beads, ticks, or microbeads.
• Antibodies can also be attached to a ProteinChip array or a probe substrate as described above.
• Detectable labels for the identification of antibodies bound to polypeptides of the technology include, but are not limited to, fluorochromes, fluorescent dyes, radiolabels, enzymes such as horse radish peroxide, alkaline phosphatase and others commonly used in the art, and colorimetric labels including colloidal gold or coloured glass or plastic beads.
• the antibody can be detected using an indirect assay, wherein, for example, a second, labelled antibody is used to detect bound polypeptide-specific antibody.
• Methods for detecting the presence of, or measuring the amount of, an antibodymarker complex include, for example, detection of fluorescence, chemiluminescence, luminescence, absorbance, birefringence, transmittance, reflectance, or refractive index such as surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler wave guide method, or
• Radio frequency methods include multipolar resonance spectroscopy.
• Electrochemical methods include amperometry and voltametry methods.
• Optical methods include imaging methods and non-imaging methods and microscopy.
• Useful assays for detecting the presence of, or measuring the amount of, an antibody-marker complex include, include, for example, enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), or a Western blot assay.
• ELISA enzyme-linked immunosorbent assay
• RIA radioimmune assay
• Western blot assay Such methods are described in, for example, Stites & Terr, (Eds) (1991) Clinical Immunology, 7th ed; and Asai, (Ed) (1993) Methods in Cell Biology : Antibodies in Cell Biology, volume 37.
• the technology provides methods for the detection and/or isolation of polynucleotides of the technology and/or polypeptides of the technology
• the technology provides a method for detecting a dinoflagellate in a sample.
• the method comprises obtaining a sample for use in the method, and detecting the presence of a polynucleotide of the technology and/or a polypeptide of the technology, or a fragment or variant thereof in the sample.
• the presence of the polynucleotide, polypeptide, or variant or fragment thereof in the sample is indicative of dinoflagellates in the sample.
• the present inventors have determined that the sxtG gene is present in saxitoxin-producing dinoflagellates and that absence of sxtG indicates dinoflagellates that do not produce saxitoxin. Accordingly, in another
• the technology provides a method for detecting a saxitoxin- producing dinoflagellate in a sample.
• the method comprises obtaining a sample for use in the method, and detecting the presence of a polynucleotide of the technology and/or a polypeptide of the technology, or a fragment or variant thereof in the sample.
• the presence of the polynucleotide, polypeptide, or variant or fragment thereof in the sample is indicative of a saxitoxin-producing dinoflagellate in the sample.
• the technology provides a method for determining an absence of saxitoxin-producing dinoflagellates in a sample.
• the method comprises obtaining a sample for use in the method, and determining an absence of a polynucleotide of the technology and/or a polypeptide of the technology, or a fragment or variant thereof in the sample.
• the absence of the polynucleotide, polypeptide, or variant or fragment thereof in the sample is indicative that saxitoxin-producing dinoflagellates are not present in the sample.
• the polynucleotide sequence may be a saxitoxin A gene (sxtA) sequence.
• the sxtA polynucleotide sequence may comprise any one or more sxtA gene catalytic domain(s) (e.g., the sxtAl, sxtA2, sxtA3, or sxtA4 catalytic domain(s)), or fragment(s) thereof.
• the sxtA polynucleotide sequence comprises an sxtAl and/or a sxtA4 domain, or fragment(s) thereof. More preferably, the sxtA polynucleotide sequence comprises an sxtA4 domain, or fragment(s) thereof.
• the polypeptide sequence may be saxitoxin A polypeptide (SXTA) sequence.
• the polynucleotide sequence may be a saxitoxin G gene ⁇ sxtG ⁇ sequence.
• the sxtG polynucleotide sequence may comprise any one or more sxtG gene catalytic domain(s) or fragment(s) thereof.
• the nucleotide sequence corresponds to the sequence provided in Figure 1 (e.g., SEQ ID NOs: 12 and/or 13.
• the technology comprises detecting both sxtA and sxtG sequences, e.g., using the primers in Table 1 (e.g., SEQ ID NOs : 1-9) and the primers defined as having the sequences defined by SEQ ID NOs : 10 and 11.
• the polypeptide sequence may be a saxitoxin A protein (SXTA) sequence.
• the SXTA polypeptide sequence may comprise any one or more SXTA protein catalytic domain(s) (e.g., the SXTAl, SXTA2, SXTA3, or SXTA4 catalytic domain(s)), or fragment(s) thereof.
• the SXTA polypeptide sequence comprises an SXTAl or an SXTA4 domain, or fragment(s) thereof. More preferably, the SXTA polypeptide sequence comprises an SXTA4 domain, or fragment(s) thereof.
• the polypeptide sequence may be a saxitoxin G protein (SXTG) sequence.
• SXTG polypeptide sequence may comprise any one or more SXTG protein catalytic domain(s) or fragment(s) thereof.
• Dinoflagellates detected in a sample or determined to be absent from a sample using the methods of the technology may be saxitoxin-producing dinoflagellates.
• the dinoflagellates may be from the order Gonyaulacales or Gymnodiniales.
• the dinoflagellates may be from the genus Alexandrium (formerly Gonyaulax) , Pyrodinium, or
• Alexandrium species include A. catenella (e.g. strains ACCCOl, ACSH02, ACTRA02 and CCMP1493), A. fundyense (e.g. strains CCMP1719 and CCMP1979), A. lusitanicum, A. minutum (e.g. strains CCMP1888, CCMP113, ALSPOl, ALSP02 and AMD 16/AMAD 16) , A. ostenfeldii, and A. tamarense (e.g. strains CCMP1771, ATBB01, ATEB01, ATCJ33 and
• Gymnodinium species include G. catenatum (e.g. strains GCTRAOl and CS-395).
• Pyrodinium species include P. bahamense var compressum.
• a sample for use in the methods of the technology may be "obtained” by any means.
• the sample may be obtained by removing it from a naturally-occurring state (e.g. a sample from a lake, ocean or river), or, by removing it from a "non-natural” state (e.g. a culture in a laboratory setting, dam, reservoir, tank etc.).
• a naturally-occurring state e.g. a sample from a lake, ocean or river
• a “non-natural” state e.g. a culture in a laboratory setting, dam, reservoir, tank etc.
• a sample for use in the methods of the technology may be suspected of comprising one or more dinoflagellates, or one or more saxitoxin-producing dinoflagellates.
• the sample may be a comparative or control sample, for example, a sample comprising a known concentration or density of
• a sample may be an environmental sample.
• the environmental sample may be derived from, for example, saltwater, freshwater, a river, a lake, an ocean, or coastal waters.
• the environmental sample may be derived from a dinoflagellate bloom.
• the sample may be derived from a laboratory source, such as a culture, or a commercial source.
• the sample may be derived from a biological source such as, for example, tissue or biological fluid.
• the sample may be modified from its original state, for example, by purification, dilution or the addition of any other component or components.
• a sample tested using the methods of the technology may provide information regarding the presence or absence of saxitoxin in animals populating the source of the sample.
• the sample may be tested to determine the presence or absence of saxitoxin in animal seafoods such as, for example, fish (e.g., pufferfish) and in particular shellfish (e.g. mussels, clams, oysters, scallops and the like).
• Polynucleotides and polypeptides for use in methods of the technology may be isolated (e.g., extracted) from microorganisms either in mixed culture or as individual species or genus isolates. Accordingly, the microorganisms of a sample may be cultured prior to extraction or the extraction may be performed directly on a given sample. Suitable methods for the isolation (e.g., extraction) and purification of polynucleotides and polypeptides for analysis using methods of the technology are generally known in the art and are described, for example, in standard texts such as Ausubel (Eds) Current Protocols in Molecular Biology (2007), John Wiley and Sons, Inc! Coligan et al.
• methods of the technology may be performed without isolating nucleic acids and/or polypeptides from the sample.
• Detecting the presence (or determining the absence) of polynucleotides of the technology and/or polypeptides of the technology in a given sample may be performed using any suitable technique.
• Suitable techniques may typically involve the use of a primer, probe or antibody specific for any one or more polynucleotides of the technology or any one or more polypeptides of the technology.
• Suitable techniques include, for example, the polymerase chain reaction (PCR) and related variations of this technique (e.g. quantitative PCR), antibody based assays such as ELISA, western blotting, flow cytometry, fluorescent microscopy, and the like.
• PCR polymerase chain reaction
• antibody based assays such as ELISA, western blotting, flow cytometry, fluorescent microscopy, and the like.
• detecting the presence (or determining the absence) of polynucleotides of the technology in a given sample is achieved by amplification of nucleic acids extracted from a sample of interest by polymerase chain reaction using primers that hybridise specifically to the polynucleotide sequence, and detecting the amplified sequence.
• oligonucleotide primers can be designed for use in PCR reactions to amplify polynucleotides of the technology such as, for example, RNA (e.g. mRNA), DNA and/or cDNA polynucleotides.
• Suitable methods of PCR include, but are not limited to, those using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector- specific primers, partially- mismatched primers, and the like.
• Methods for designing PCR and RT-PCR primers are generally known in the art and are disclosed, for example, in standard texts such as Ausubel et al. (Eds) Current Protocols in Molecular Biology (2007), John Wiley and Sons, Inc! Maniatis et al. Molecular Cloning (1982), 280-281; Innis et al.
• PCR and RT-PCR procedures may be altered without affecting the ability to obtain the desired product.
• the salt concentration may be varied or the time and/or temperature of one or more of the denaturation, annealing and extension steps may be varied.
• the amount of DNA, cDNA, or RNA template may also be varied depending on the amount of nucleic acid available or the optimal amount of template required for efficient amplification.
• the primers for use in the methods and kits of the present technology are typically
• oligonucleotides typically being at least about nucleotides to about 80 nucleotides in length, more typically about 10 nucleotides in length to about 50 nucleotides in length, and even more typically about 15 nucleotides in length to about 30 nucleotides in length.
• primers of the technology may be useful for a number of different applications, including but not limited to, PCR, RT-PCR, and as probes for the detection of polynucleotides of the technology.
• Such primers can be prepared by any suitable method, including, for example, direct chemical synthesis or cloning and restriction of appropriate sequences. Not all bases in the primer need reflect the sequence of the template molecule to which the primer will hybridize. The primer need only contain sufficient complementary bases to enable the primer to hybridize to the template.
• a primer may also include mismatch bases at one or more positions, being bases that are not complementary to bases in the template, but rather are designed to incorporate changes into the DNA upon base extension or amplification.
• a primer may include additional bases, for example in the form of a restriction enzyme recognition sequence at the 5' end, to facilitate cloning of the amplified DNA.
• the methods of the technology involve detecting the presence (or determining the absence) of polynucleotides of the technology and/or
• sequences may comprise saxitoxin A sequences including any one or more of the saxitoxin Al, A2, A3 or A4 catalytic domain sequences (or fragment(s) thereof).
• sequences may comprise saxitoxin G sequences.
• sequences may comprise a sequence comprising both sxtA and sxtG, e.g., a sxtAlsxtG sequence.
• any primer(s) capable of the amplifying a polynucleotide of the technology any probe capable of detecting a polynucleotide of the technology, or any antibody capable of detecting a polypeptide of the technology, may be used when performing the methods of the technology.
• the primers, probes and antibodies bind specifically to any one or more of the saxitoxin G sequences referred to in the preceding paragraph (e.g., paragraph directly above).
• binding specifically it will be understood that the primer, probe or antibody is capable of binding to the target sequence with a higher affinity than it binds to an unrelated sequence. Accordingly, when exposed to a plurality of different but equally accessible sequences as potential binding partners, the primer, probe or antibody specific for a target sequence will selectively bind to the target sequence and other alternative potential binding partners will remain substantially unbound by the primer, probe or antibody.
• a primer, probe or antibody specific for a target sequence will preferentially bind to the target sequence at least 10-fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than to other potential sequences that are not target sequences.
• a primer, probe or antibody specific for a target sequence may be capable of binding to non-target sequences at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from specific binding, for example, by use of an appropriate control.
• Suitable primers and probes may bind specifically to any fragment of a saxitoxin G polynucleotide sequence.
• Suitable antibodies may bind specifically to a fragment of a saxitoxin G polypeptide sequence encoded by such polynucleotide sequences.
• suitable primers and probes may bind specifically to a fragment of the saxitoxin G polynucleotide sequence defined by SEQ ID NOs : 12 and/or 13 as provided herein.
• Suitable antibodies may bind specifically to a fragment of a saxitoxin G polypeptide sequence encoded by such polynucleotide sequences.
• the methods of the technology may involve detecting the presence (or determining the absence) of polynucleotides of the technology in a sample using PCR amplification.
• Suitable oligonucleotide primer pairs for the PCR amplification of saxitoxin G polynucleotide sequences may be capable of amplifying any one or more domain(s) of the sxtG gene, or
• primer pairs for this purpose may comprise primers provided in Table 1.
• Table 1 The skilled addressee will recognise that the exemplified primers are not intended to limit the region of the saxitoxin G gene amplified or the methods of the technology in general.
• the technology is not limited to the use of the specific primers exemplified, and alternative primer sequences may also be used, provided the primers are designed appropriately so as to enable the amplification of saxitoxin polynucleotide sequences, preferably saxitoxin G polynucleotide sequences, and in some embodiments, both sxtG and sxtA sequences.
• technology involves detecting the presence
• Suitable oligonucleotide primer pairs for the PCR amplification of saxitoxin A polynucleotide sequences may be capable of amplifying any one or more regions, e.g., one or more catalytic domain(s), of the sxtA gene, or fragments(s) thereof.
• the primers amplify a sequence comprising a saxitoxin A4 catalytic domain polynucleotide sequence, or a fragment thereof.
• a suitable primer pair for this purpose may comprise a first primer comprising the polynucleotide sequence defined as
• CTGAGCAAGGCGTTCAATTC SEQ ID NO: 10
• a second primer comprising the polynucleotide sequence defined as
• TACAGATMGGCCCTGTGARC (SEQ ID NO: ll), or a fragment or variant thereof.
• the exemplified primers are not intended to limit the region of the saxitoxin A gene amplified or the methods of the invention in general.
• the invention is not limited to the use of the specific primers exemplified, and alternative primer sequences may also be used, provided the primers are designed appropriately so as to enable the amplification of saxitoxin (e.g., sxtA and/or sxtCr) polynucleotide sequences.
• saxitoxin e.g., sxtA and/or sxtCr
• the methods of the technology may involve detecting the presence (or determining the absence) of polynucleotides of the technology in a sample by the use of suitable probes.
• Probes of the technology are based on sxt polynucleotide sequences of the technology. Probes are nucleotide sequences of variable length, for example between about 10 nucleotides and several thousand nucleotides, for use in detection of homologous sequences, typically by hybridization.
• Hybridization probes of the technology may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides.
• Probes of the technology may be labelled by incorporation of a marker to facilitate their detection. Examples of suitable markers include fluorescent molecules (e.g.
• acetylaminofluorene 5-bromodeoxyuridine, digoxigenin, fluorescein
• radioactive isotopes e.g. 32 P, 35 S, 3 H, 33 P.
• Detection of the marker may be achieved, for example, by chemical, photochemical, immunochemical, biochemical, or spectroscopic techniques. Methods for the design and/or production of nucleotide probes are generally known in the art, and are described, for example, in standard texts such as Robinson et al. (Eds) Current Protocols in Cytometry (2007), John Wiley and Sons, Inc! Ausubel et al. (Eds) Current Protocols in Molecular Biology (2007), John Wiley and Sons, Inc! Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York; and Maniatis et al.
• the methods of the technology may involve detecting the presence (or determining the absence) of polypeptides of the technology in a sample using antibodies.
• the antibodies may be used to qualitatively or quantitatively to detect and analyse one or more SXT
• polypeptides of the technology in a given sample.
• the antibodies may be conjugated to a fluorochrome allowing detection, for example, by flow cytometry, immunohistochemisty or other means known in the art.
• the antibody may be bound to a substrate allowing colorimetric or chemiluminescent detection.
• the technology also contemplates the use of secondary antibodies capable of binding to one or more antibodies capable of binding specifically to a polypeptide of the technology.
• the technology also provides kits for the detection and/or isolation of
• kits are used for detecting a dinoflagellate in a sample. In other embodiments the kits are used for detecting a saxitoxin- producing dinoflagellate in a sample. In other embodiments the kits are used for determining an absence of saxitoxin producing dinoflagellates in a sample.
• the kits of the technology comprise at least one agent for detecting the presence of one or more polynucleotides of the technology and/or one or more polypeptides of the technology, and/or variants or fragments thereof (see description in the section above entitled "Polynucleotides and polypeptides"). Any agent suitable for this purpose may be included in the kits. Non-limiting examples of suitable agents include primers, probes and antibodies such as those described above in the sections entitled “Probes, primers and antibodies” and "Methods for detecting dinoflagellates”.
• kits are for use in the methods of the technology (see description in the section above entitled "Methods for detecting dinoflagellates”).
• the technology provides a kit for the detection of a dinoflagellate in a sample, the kit comprising at least one agent for detecting in the sample the presence of one or more polynucleotides of the technology, and/or one or more polypeptides of the technology, and/or a variant or fragment of either.
• the dinoflagellate is a saxitoxin-producing dinoflagellate.
• the technology provides a kit for determining the absence of a dinoflagellate in a sample, the kit comprising at least one agent for determining in the sample the absence of one or more polynucleotides of the technology, and/or one or more polypeptides of the technology, and/or a variant or fragment of either.
• the dinoflagellate is a saxitoxin-producing dinoflagellate.
• kits of the technology may comprise any number of additional components.
• the additional components may include components for collecting and/or storing samples, reagents for cell culture, reference samples, buffers, labels, and/or written instructions for performing method(s) of the technology.
• Dinoflagellates detected in a sample or determined to be absent from a sample using kits of the technology may be saxitoxin-producing dinoflagellates.
• the dinoflagellates may be from the order Gonyaulacales or Gymnodiniales.
• the dinoflagellates may be from the genus Alexandrium (formerly Gonyaulax), Pyrodinium, or Gymnodinium. Suitable examples of Alexandrium species include A. catenella (e.g. strains ACCCOl, ACSH02, ACTRA02 and CCMP1493), A.
• fundyense e.g. strains CCMP1719 and CCMP1979
• A. lusitanicum e.g. strains CCMP1888, CCMP113, ALSP01, ALSP02 and AMD 16/AM AD 16
• A. ostenfeldii e.g. strains CCMP1771, ATBB01, ATEB01
• A. tamarense e.g. strains CCMP1771, ATBB01, ATEB01
• Gymnodinium species include G. catenatum (e.g. strains GCTRAOl and CS-395). Suitable examples of
• Pyrodinium species include P. bahamense var compressum. It will be
• dinoflagellates were cultured and tested.
• the dinoflagellate species/strains used in this study included Adenoides eludens CCMP1891, Alexandrium affine CCMP112, Alexandrium andersoni CCMP2222, Alexandrium catenella
• CCMP1771 Amphidinium carter/ UIO081, Amphidinium massartii CS-259, Amphidinium mootonorum CAWD161, Azadinium spinosum RCC2538,
• chrysophyte Phycologia 32: 234-236) at 16-25°C, and Polarella glacialis CCMP2088 was grown at 5°C. All strains were grown with a 12 hour/12 hour light-dark photoperiod and a photon irradiance of ⁇ 100 mmol photons m "2 s "1 . Strains were not maintained axenic. Culture identity was confirmed with a 18S PCR using the NSF83 and 1528R primers (see, e.g., Hendriks L, et al. (1989), "The Nucleotide-Sequence of the Small Ribosomal-Subunit RNA of the Yeast Candida albicans and the Evolutionary Position of the Fungi among the
• Candidate hits (E-value ⁇ 0.1) were identified using a custom BLAST against the cyanobacterial SxtG amino acid sequence, before being re-assembled with MacClade v4.07 (Sinauer Associates). Resulting contigs were BLASTed against the non-redundant EST and SRA databases at NCBI. Orthologous Alexandrium EST (Accessions: EX463008, CK786100, and CK782453) and SRA (Accession: SRX111568) sequences were identified and aligned with all sxtG sequences before designing primers (as discussed below).
• the sxtG transcript was amplified by various primer methods (see Table 1 for primer sequences).
• the primer pair sxtGl60F and sxtGl005R and negative amplifications were used (l ⁇ product as template) in a nested system with the primer pair sxtG203F and sxtGl005R.
• a nested system using the Zhang dinoSL Zhang H, et al. (2007), "Spliced leader RNA trans- splicing in dinoflagellates", PNAS IO '- 4618-4623
• AUAP Invitrogen
• the 5' end of the transcript, including the spliced leader sequence was amplified using the dinoSL primer and the sxtg660R primer.
• the 3' end was amplified using the sxtg203F and the AUAP adaptor primer.
• SxtG presence was checked for all dinoflagellates (genomic and transcript) with the primer pair sxtG203F and sxtG660R as well as with the nested system, sxtG203F+sxtGl005R / sxtGl60F+sxtGl005R.
• PCR was performed with 3% DMSO with the following conditions 1 an initial 5 minute 95°C denaturing before 35 cycles of (l) 30 seconds at 94°C (denaturing); (2) 30 seconds at 62°C (annealing); and (3) 1-2 minutes at 72°C (extension), with a final 10- minute extension at the same temperature. All other PCR parameters were standard as described below.
• the 5' and 3' end products were cloned with TOPO TA (Invitrogen) before being sequenced with the M13 forward and reverse primers. All other PCR products were directly sequenced with respective forward and reverse primer, as well as several internal primers covering the entire transcript. Sequences were assembled as discussed below.
• Primer name Primer direction Primer sequence 5' -3' SEQ ID NO:
• Genomic DNA and Total RNA was isolated from 20 ml of culture in the exponential growth phase, centrifuged for 2 minutes at 12,000 x g, washed with PBS, and bead-beaten on dry ice with the FastPrep-24 from Medinor (20 seconds, speed 4) using 1.4-mm beads (Medinor). Then, the samples were processed with the Invitrogen ChargeSwitch gDNA plant kit (Invitrogen) and Invitrogen
• RNA cell kit ChargeSwitch TotalRNA cell kit (Invitrogen) in accordance with supplied protocol.
• Total RNA from Gymnodinium catenatum (CCMP1937) was kindly donated by Johannes Hagstrom.
• First strand cDNA was synthesized with the Invitrogen 3' RACE system (Invitrogen) following the high-GC protocol and utilizing the (AP) adapter primer. DNA, RNA, and cDNA quality was checked with a NanoDrop spectrophotometer (ThermoScientific).
• PCR Using PCR, template was amplified using Qiagen HotStarTaq Plus polymerase (Qiagen) in the presence of 10% BSA in a MJ Research PTC-200 Thermo Cycler (MJ Research). PCR products were gel excised using Promega Wizard SV Gel and PCR Clean-Up System (Promega) before direct sequencing with an ABI3730 DNA analyzer (Applied Biosystems). Primers used in this study have been designed using Primaclade ⁇ Bioinformatics 21 : 1263-1264). Melting temperature (TM) was calculated using OligoCalc ⁇ Nucleic Acids
• cyanobacteria Applied and Environmental Microbiology! '4- 4044-4053.
• the dinoflagellate sxtG amino acid sequences were aligned using MAFFTv6 L- INS-I model (Katoh K and Toh H (2008) "Improved accuracy of multiple ncRNA alignment by incorporating structural information into a MAFFT-based framework", BMC Bioinformatics 9- 13) to orthologous cyanobacteria sxtG sequences, in addition to a selection of closely related NCBInr Blastp hits.
• PCR amplification was used to amplify sxtG and sxtA from DNA template prepared from A. affine (CCMP112), A. andersoni (CCMP2222), A. catenella (CCMP1493), A. insuetum (CCMP2082), A. tamarense (CCMP1771), A. carter! (UIO081), C longipes (CCMP1770), C monotis, G catenatum (CCMP1937), H. triquetra (RCC2540), K. veneficum (RCC2539), L. polyedrum (CCMP1931), P. lima (CS-869), P. micans (UI0292), P. minimum (UIO085), P. noctiluca
• CCMP732 CCMP732
• P. reticulatum P. reticulatum
• sxtG transcripts for both A. fundyense CCMP1719 and A. minutum CCMP113. This included dinoflagellate spliced- leader sequence at the 5' end and a eukaryotic poly-A tail at the 3' end. The transcripts were 1283 bp and 1276 bp in length, respectively, excluding the poly- A tails. Additionally, the A. tamarense SRA contig (SRX111568) contained 9 bp of the 22 bp dinoSL sequence. conserveed domain searches identified sxtG as an amidinotransferase. The ORF was predicted to be 375 amino acids in length.
• the 1125 bp ORF contig of A fundyense CCMP1719 had 5 single nucleotide polymorphisms (SNPs) all of which are non-synonymous. In comparison the same region of A minutum CCMP113 lacked a single SNP.
• SNPs single nucleotide polymorphisms
• the sxtG primers designed in this study amplified an 881 bp sequence from seven Alexandrium species (both STX producers and non-producers) and Gymnodinium catenatum (Table 2). No sxtGVCR products were amplified for 23 non-STX-producing dinoflagellates (Table 2). In addition, no putative sxtG sequence external to Alexandrium was identified by BLAST against the NCBI non-redundant, EST and SRA databases.
• the phylogenetic inference of sxtG shows that dinoflagellate sxtG sequences form a fully supported clade.
• the planctomycete Gemmata obscuriglobus is the unsupported sister to the dinoflagellate sxtG clade.
• the proteobacterium Beggiatoa further excludes the dinoflagellate sequences from a sister relationship with a fully supported cyanobacterial sxtG clade.
• the four previous clades form a weakly supported (61/0.66) group with a cluster of proteobacteria species. This is further included in a moderately supported (76/0.98) monophyly with an additional proteobacteria clade, constituting the cluster defined amidinotransferase 1.
• amidinotransferase 1 clade is excluded from Opisthokonta (100/1.00) and amidinotransferase 2 (92/0.99).
• the amidinotransferase 2 clade harbors the additional dinoflagellate amidinotransferase sequences. This fully supported clade forms a weak (53/0.82) grouping to Actinobacteria and Cyanobacteria AoaA sequences.

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