Detection of toxigenic marine diatoms of the genus Pseudo-nitzschia
AssigneeMonterey Bay Aquarium Research Institute
InventorsChristopher A. Scholin, Gerard A. Cangelosi, Paul V. Haydock
Life of this patent
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Abstract
The present invention provides compositions, methods, and kits for detecting species of Pseudo-nitzschia from a marine sample. Oligonucleotide probes for rRNA hypervariable regions of the Psuedo-nitzschia species: P. australis, P. pungens, P. multiseries, P. pseudodelicatissima, P. heimii, P. fraudulenta, P. delicatissima, and P. americana are provided as well as a oligonucleotide probe for a conserved region of ribosomal RNA from Pseudo-nitzschia.
Claims23
A composition of polynucleotide probes for the detection of at least one Pseudo-nitzschia species from a marine sample, wherein said probes comprise a segment of nucleic acid capable of selectively hybridizing, under selective hybridizing conditions, to a hypervariable region of ribosomal RNA selected from at least one of the group consisting of:
- for P. australis
- 5'-CCGCCUGGUGGAGUGAGUCAUUU-3' (SEQ ID NO:1)
- 5'-CCUGGUGGAGUGAGUCAUUU-3' (SEQ ID NO:2)
- for P. pungens
- 5'-CCGCCUGGUAAAGUGAGUCA-3' (SEQ ID NO:3)
- 5'-UGGUAAAGUGAGUCAU-3' (SEQ ID NO:4)
- 5'-GGCCUGGGCGCUGUGAGCUU-3' (SEQ ID NO:5)
- for P. multiseries
- 5'-CCUGGCAGAGUGAGUCAUUU-3' (SEQ ID NO:6)
- 5'-UGGCAGAGUGAGUCAU-3' ( SEQ ID NO:7)
- for P. multiseries and P. pseudodelicatissima
- 5'-UGGCUUGGGCGCUGUGGGCU-3' (SEQ ID NO:8)
- 5'-GGCUUGGGCGCUGUGGGCUU-3' (SEQ ID NO:9)
- for P. heimii
- 5'-UCUGGUAGAAUGAGUCAUGG-3' (SEQ ID NO:10)
- 5'-UAUGUUCAUAUUUCCCUUG-3' (SEQ ID NO:11)
- 5'-UGUGGGCGCUGUGGAUA-3' (SEQ ID NO:12)
- for P. fraudulenta
- 5'-CCUGGUAGAAUGAGUCUUU-3' (SEQ ID NO:13)
- for P. delicatissima
- 5'-UGGUAGAGUGAGUCU-3' (SEQ ID NO:
- 14)
- for P. americana
- 5'-UGGCUGAAUGAGUCAU-3' (SEQ ID NO:15);
- with the proviso that any additional nucleic acid sequences covalently bound to said segment do not hybridize under said conditions to nucleic acids of Pseudo-nitzschia species in said sample.
A polynucleotide probe for the detection of Pseudo-nitzschia species from a marine sample, wherein said probe comprises a segment of nucleic acid capable of selectively hybridizing, under selective hybridizing conditions, to a conserved region of ribosomal RNA, wherein said conserved region is:
- 5'-GATGCAAAGAACUUUGAAAAGAGAGUUAAAGAG-3' (SEQ ID NO:16)
- with the proviso that any additional nucleic acid sequences covalently bound to said segment do not hybridize under said conditions to nucleic acids of Pseudo-nitzschia species in said sample.
A method of detecting at least one Pseudo-nitzschia species from a marine sample, said method comprising the steps of:
- permeabilizing the Pseudo-nitzschia to expose ribosomal RNA;
- contacting said ribosomal RNA, under hybridizing conditions, with polynucleotide probes comprising a segment of nucleic acid capable of selectively hybridizing, under selective hybridizing conditions, to a hypervariable region of ribosomal RNA of said Pseudo-nitzschia wherein said hypervariable region is selected from at least one of the group consisting of:
- for P. australis
- 5'-CCGCCUGGUGGAGUGAGUCAUUU-3' (SEQ ID NO:1)
- 5'-CCUGGUGGAGUGAGUCAUUU-3' (SEQ ID NO:2)
- for P. pungens
- 5'-CCGCCUGGUAAAGUGAGUCA-3' (SEQ ID NO:3)
- 5'-UGGUAAAGUGAGUCAU-3' (SEQ ID NO:4)
- 5'-GGCCUGGGCGCUGUGAGCUU-3' (SEQ ID NO:5)
- for P. multiseries
- 5'-CCUGGCAGAGUGAGUCAUUU-3' (SEQ ID NO:6)
- 5'-UGGCAGAGUGAGUCAU-3' (SEQ ID NO:7)
- for P. multiseries and P. pseudodelicatissima
- 5'-UGGCUUGGGCGCUGUGGGCU-3' (SEQ ID NO:8)
- 5'-GGCUUGGGCGCUGUGGGCUU-3' (SEQ ID NO:9)
- for P. heimii
- 5'-UCUGGUAGAAUGAGUCAUGG-3' (SEQ ID NO:10)
- 5'-UAUGUUCAUAUUUCCCUUG-3' (SEQ ID NO:11)
- 5'-UGUGGGCGCUGUGGAUA-3' (SEQ ID NO:
- 12)
- for P. fraudulenta
- 5'-CCUGGUAGAAUGAGUCUUU-3' (SEQ ID NO:
- 13)
- for P. delicatissima
- 5'-UGGUAGAGUGAGUCU-3' (SEQ ID NO:14)
- for P. americana
- 5'-UGGCUGAAUGAGUCAU-3' (SEQ ID NO:
- 15);
- and detecting hybridization complexes as an indication of the presence of Pseudo-nitzschia species in said sample, with the proviso that any additional nucleic acid sequences covalently bound to said segment do not hybridize under said conditions to nucleic acids of Pseudo-nitzschia species in said sample.
A diagnostic kit for use in determining the presence of Pseudo-nitzschia in a marine sample which comprises in a container a synthetic oligonucleotide probe comprising a segment of nucleic complementary to a hypervariable region of ribosomal RNA of Pseudo-nitzschia selected from the group consisting of:
- for P. australis
- 5'-CCGCCUGGUGGAGUGAGUCAUUU-3' (SEQ ID NO:1)
- 5'-CCUGGUGGAGUGAGUCAUUU-3' (SEQ ID NO:2)
- for P. pungens
- 5'-CCGCCUGGUAAAGUGAGUCA-3' (SEQ ID NO:3)
- 5'-UGGUAAAGUGAGUCAU-3' (SEQ ID NO:4)
- 5'-GGCCUGGGCGCUGUGAGCUU-3' (SEQ ID NO:5)
- for P. multiseries
- 5'-CCUGGCAGAGUGAGUCAUUU-3' (SEQ ID NO:6)
- 5'-UGGCAGAGUGAGUCAU-3' (SEQ ID NO:7)
- for P. multiseries and P. pseudodelicatissima
- 5'-UGGCUUGGGCGCUGUGGGCU-3' (SEQ ID NO:8)
- 5'-GGCUUGGGCGCUGUGGGCUU-3' (SEQ ID NO:9)
- for P. heimii
- 5'-UCUGGUAGAAUGAGUCAUGG-3' (SEQ ID NO:10)
- 5'-UAUGUUCAUAUUUCCCUUG-3' (SEQ ID NO:11)
- 5'-UGUGGGCGCUGUGGAUA-3' (SEQ ID NO:12)
- for P. fraudulenta
- 5'-CCUGGUAGAAUGAGUCUUU-3' (SEQ ID NO:13)
- for P. delicatissima
- 5'-UGGUAGAGUGAGUCU-3' (SEQ ID NO:14)
- for P. americana
- ' -UGGCUGAAUGAGUCAU-3' (SEQ ID NO:15)
- and combinations thereof, with the proviso that any additional nucleic acid sequences covalently bound to said segment do not hybridize under said conditions to nucleic acids of Pseudo-nitzschia species in said sample.
Dependencies are inferred from the claim text.