USE OF PROBES TO DETECT TOXINOGENIC CYANOBACTERIA, DETECTION METHOD AND CORRESPONDING KITS

Abstract

Probes for the detection of toxinogenic cyanobacteria, and the use of at least one pair of these probes in a method for the detection of the toxinogenic cyanobacteria in a sample likely to contain the toxinogenic cyanobacteria. Also, corresponding kits including at least one pair probes specific to toxinogenic cyanobacteria.

Claims

1-21. (canceled)

22. A method for detecting toxinogenic cyanobacteria in a sample likely to contain at least one toxinogenic cyanobacteria of the genus Microcystis using at least one pair of probes specific to toxinogenic cyanobacteria of the genus Microcystis, the sequences of the said probes being chosen from x elements of one of the following sets: (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4) (SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7) (SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11) (SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14) (SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17) (SEQ ID NO: 18 and SEQ ID NO: 19) x being 2, 3 or 4 or the sequences of said probes having at least 92% identity with said sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, one probe of said pair being a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ of its sequence and the other probe of said pair being a signal probe linked to at least one marking molecule positioned at 3′ or 5′ of its sequence, said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of the toxinogenic cyanobacteria of the genus Microcystis optionally present in said sample to form a complex, the minimum detection threshold of the toxinogenic cyanobacteria of the genus Microcystis being estimated from 10 to 575 active living cells per millilitre of sample (cells/mL), or less than or equal to 1.0 ng of ribosomal RNA per millilitre of sample, and in particular from 0.02 ng to 0.7 ng of ribosomal RNA per millilitre of sample and preferably from 0.02 ng to 0.1 ng of ribosomal RNA per millilitre of sample, in particular, the sequences of the probes of the said pairs being as follows: (SEQ ID NO: 1 and SEQ ID NO: 2), (SEQ ID NO: 1 and SEQ ID NO: 3), (SEQ ID NO: 1 and SEQ ID NO: 4), (SEQ ID NO: 2 and SEQ ID NO: 3), (SEQ ID NO: 3 and SEQ ID NO: 4) (SEQ ID NO: 5 and SEQ ID NO: 6), (SEQ ID NO: 5 and SEQ ID NO: 7), (SEQ ID NO: 6 and SEQ ID NO: 7) (SEQ ID NO: 8 and SEQ ID NO: 9), (SEQ ID NO: 8 and SEQ ID NO: 10), (SEQ ID NO: 8 and SEQ ID NO: 11), (SEQ ID NO: 9 and SEQ ID NO: 10), (SEQ ID NO: 9 and SEQ ID NO: 11), (SEQ ID NO: 10 and SEQ ID NO: 11) (SEQ ID NO: 12 and SEQ ID NO: 13), (SEQ ID NO: 12 and SEQ ID NO: 14), (SEQ ID NO: 13 and SEQ ID NO: 14) (SEQ ID NO: 15 and SEQ ID NO: 16), (SEQ ID NO: 15 and SEQ ID NO: 17), (SEQ ID NO: 16 and SEQ ID NO: 17) (SEQ ID NO: 18 and SEQ ID NO: 19).

23. The method according to claim 22, for detecting toxinogenic cyanobacteria in a sample likely to contain at least one of toxinogenic cyanobacteria of the genus Microcystis and/or Aphanizomenon, using, in addition, at least one pair of probes specific to toxinogenic cyanobacteria of the genus Aphanizomenon, the sequences of said probes being chosen from x elements of one of the following sets: (SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22) (SEQ ID NO: 23 and SEQ ID NO: 24) x being 2 or 3, or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, one probe of said pair being a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ of its sequence and the other probe of said pair being a signal probe linked to at least one marking molecule positioned at 3′ or 5′ of its sequence said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Aphanizomenon optionally present in said sample to form a complex, the minimum detection threshold for the toxinogenic cyanobacteria of the genus Aphanizomenon being estimated from 10 to 575 active living cells per millilitre of sample (cells/mL), or less than or equal to 1.0 ng of ribosomal RNA per millilitre of sample, and in particular from 0.02 ng to 0.7 ng of ribosomal RNA per millilitre of sample and preferably from 0.02 ng to 0.1 ng of ribosomal RNA per millilitre of sample, in particular, the sequences of the probes of the said pairs being as follows: (SEQ ID NO: 20 and SEQ ID NO: 21), (SEQ ID NO: 20 and SEQ ID NO: 22), (SEQ ID NO: 21 and SEQ ID NO: 21) (SEQ ID NO: 23 and SEQ ID NO: 24).

24. The method according to claim 22, for detecting toxinogenic cyanobacteria, in a sample likely to contain at least one toxinogenic cyanobacteria of the genus Microcystis and/or Dolichospermum, using in addition at least one pair of probes specific to toxinogenic cyanobacteria of the genus Dolichospermum, the sequences of said probes being chosen from x elements of one of the following sets: (SEQ ID NO: 25 and SEQ ID NO: 26) x being 2, or the sequences of said probes having at least 92% identity with the aforementioned sequences SEQ ID NO: 25, SEQ ID NO: 26, one probe of said pair being a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ of its sequence and the other probe of said pair being a signal probe linked to at least one marking molecule positioned at 3′ or 5′ of its sequence said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Dolichospermum optionally present in said sample to form a complex, the minimum detection threshold for the toxinogenic cyanobacteria of the genus Dolichospermum being estimated from 10 to 575 active living cells per millilitre of sample (cells/mL), or less than or equal to 1.0 ng of ribosomal RNA per millilitre of sample, and in particular from 0.02 ng to 0.7 ng of ribosomal RNA per millilitre of sample and preferably from 0.02 ng to 0.1 ng of ribosomal RNA per millilitre of sample.

25. The method according to claim 22, for detecting toxinogenic cyanobacteria, in a sample likely to contain at least one toxinogenic cyanobacteria of the genus Microcystis and/or Anabaena, using in addition at least one pair of probes specific to toxinogenic cyanobacteria of the genus Anabaena, the sequences of said probes being selected from x elements of one of the following sets: (SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29) (SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32) (SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35) (SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39) (SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42) (SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45) (SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48) (SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51) (SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54) (SEQ ID NO: 55 and SEQ ID NO: 56) (SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59) (SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62) x being 2, 3 or 4, or the sequences of said probes having at least 92% identity with the aforementioned sequences SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, one probe of said pair being a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ of its sequence and the other probe of said pair being a signal probe linked to at least one marking molecule positioned at 3′ or 5′ of its sequence, said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Anabaena optionally present in said sample to form a complex, the minimum detection threshold of the toxinogenic cyanobacteria of the genus Anabaena being estimated from 10 to 575 active living cells per millilitre of sample (cells/mL), or less than or equal to 1.0 ng of ribosomal RNA per millilitre of sample, and in particular from 0.02 ng to 0.7 ng of ribosomal RNA per millilitre of sample and preferably from 0.02 ng to 0.1 ng of ribosomal RNA per millilitre of sample, in particular, the sequences of the probes of the said pairs being as follows: (SEQ ID NO: 27 and SEQ ID NO: 28), (SEQ ID NO: 27 and SEQ ID NO: 29), (SEQ ID NO: 28 and SEQ ID NO: 29) (SEQ ID NO: 30 and SEQ ID NO: 31), (SEQ ID NO: 30 and SEQ ID NO: 32), (SEQ ID NO: 31 and SEQ ID NO: 32) (SEQ ID NO: 33 and SEQ ID NO: 34), (SEQ ID NO: 33 and SEQ ID NO: 35), (SEQ ID NO: 34 and SEQ ID NO: 35) (SEQ ID NO: 36 and SEQ ID NO: 37), (SEQ ID NO: 36 and SEQ ID NO: 38), (SEQ ID NO: 36 and SEQ ID NO: 39), (SEQ ID NO: 37 and SEQ ID NO: 38), (SEQ ID NO: 37 and SEQ ID NO: 39), (SEQ ID NO: 38 and SEQ ID NO: 39) (SEQ ID NO: 40 and SEQ ID NO: 41), (SEQ ID NO: 40 and SEQ ID NO: 42), (SEQ ID NO: 41 and SEQ ID NO: 42) (SEQ ID NO: 43 and SEQ ID NO: 44), (SEQ ID NO: 43 and SEQ ID NO: 45), (SEQ ID NO: 44 and SEQ ID NO: 45) (SEQ ID NO: 46 and SEQ ID NO: 47), (SEQ ID NO: 46 and SEQ ID NO: 48), (SEQ ID NO: 47 and SEQ ID NO: 48) (SEQ ID NO: 49 and SEQ ID NO: 50), (SEQ ID NO: 49 and SEQ ID NO: 51), (SEQ ID NO: 50 and SEQ ID NO: 51) (SEQ ID NO: 52 and SEQ ID NO: 53), (SEQ ID NO: 52 and SEQ ID NO: 54), (SEQ ID NO: 53 and SEQ ID NO: 54) (SEQ ID NO: 55 and SEQ ID NO: 56) (SEQ ID NO: 57 and SEQ ID NO: 58), (SEQ ID NO: 57 and SEQ ID NO: 59), (SEQ ID NO: 58 and SEQ ID NO: 59) (SEQ ID NO: 60 and SEQ ID NO: 61), (SEQ ID NO: 60 and SEQ ID NO: 62), (SEQ ID NO: 61 and SEQ ID NO: 62).

26. The method according to claim 22, for detecting toxinogenic cyanobacteria, in a sample likely to contain at least one toxinogenic cyanobacteria of the genus Microcystis and/or Planktothrix, using in addition at least one pair of probes specific to toxinogenic cyanobacteria of the genus Planktothrix, the sequences of said probes being chosen from x elements of one of the following sets: (SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66) (SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69) x being 3 or 4, or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, one probe of said pair being a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ of its sequence and the other probe of said pair being a signal probe linked to at least one marking molecule positioned at 3′ or 5′ of its sequence, said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Planktothrix optionally present in said sample to form a complex, the minimum detection threshold of the toxinogenic cyanobacteria of the genus Planktothrix being estimated from 10 to 575 active living cells per millilitre of sample (cells/mL), or less than or equal to 1.0 ng of ribosomal RNA per millilitre of sample, and in particular from 0.02 ng to 0.7 ng of ribosomal RNA per millilitre of sample and preferably from 0.02 ng to 0.1 ng of ribosomal RNA per millilitre of sample, in particular, the sequences of the probes of the said pairs being as follows: (SEQ ID NO: 63 and SEQ ID NO: 64), (SEQ ID NO: 63 and SEQ ID NO: 65), (SEQ ID NO: 63 and SEQ ID NO: 66), (SEQ ID NO: 64 and SEQ ID NO: 65), (SEQ ID NO: 64 and SEQ ID NO: 66), (SEQ ID NO: 65 and SEQ ID NO: 66) (SEQ ID NO: 67 and SEQ ID NO: 68), (SEQ ID NO: 67 and SEQ ID NO: 69), (SEQ ID NO: 68 and SEQ ID NO: 69).

27. The use according to claim 22, wherein, said capture probe is linked to at least one attachment molecule positioned 5′ to its sequence and said signal probe is linked to at least one marking molecule positioned 5′ to its sequence, or said capture probe is linked to at least one attachment molecule positioned 5′ of its sequence and said signal probe is linked to at least one marking molecule positioned 3′ of its sequence, or said capture probe is linked to at least one attachment molecule positioned 3′ of its sequence and said signal probe is linked to at least one marking molecule positioned 5′ of its sequence, or said capture probe is linked to at least one attachment molecule positioned 3′ of its sequence and said signal probe is linked to at least one marking molecule positioned 3′ of its sequence, said at least one attachment molecule being in particular selected from a biotin, avidin, streptavidin, a thiol group, an amine group and a carbon, preferably a biotin molecule, the said at least one marking molecule being chosen in particular from a fluorochrome, a biotin, a biotin-bound molecule, digoxigenin, an enzyme using a chemiluminescent substrate, an enzyme using a chromogenic substrate or an enzyme using an electrochemically oxidized substrate, preferably digoxigenin, preferably, said enzyme using a chromogenic substrate is alkaline phosphatase and said chromogenic substrate is Tetrazolium Nitroblue (NBT) or Bromochlorylindolophosphate (BCIP), or said enzyme using a chromogenic substrate is horseradish peroxidase (HRP) and said chromogenic substrate is selected from 3,3′-Diaminobenzidine (DAB), 3,3′,5,5′-Tetramethylbenzidine (TMB), or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS).

28. A method according to claim 22 for the detection of toxinogenic cyanobacteria in a sample likely to contain at least one toxinogenic cyanobacteria of the genus Microcystis comprising the following steps: a) optional hybridization resulting from the contact of the said sample with a capture probe and a signal probe specific to toxinogenic cyanobacteria of the genus Microcystis, the capture probe and the signal probe forming a pair of probes, the sequences of the said pair of probes being chosen from x elements of one of the following sets: (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4) (SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7) (SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11) (SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14) (SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17) (SEQ ID NO: 18 and SEQ ID NO: 19) x being 2, 3 or 4 or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Microcystis optionally present in said sample to form a complex, b) detection of said optional complex, hybridization indicating the presence of toxinogenic cyanobacteria of the genus Microcystis, preferably, the minimum detection threshold of the toxinogenic cyanobacteria of the genus Microcystis being estimated from 10 to 575 active living cells per millilitre of sample (cells/mL), or less than or equal to 1.0 ng of ribosomal RNA per millilitre of sample, and in particular from 0.02 ng to 0.7 ng of ribosomal RNA per millilitre of sample and preferably from 0.02 ng to 0.1 ng of ribosomal RNA per millilitre of sample, preferably, the duration of the implementation of the said detection method being less than one hour.

29. The method for the detection of toxinogenic cyanobacteria according to any one of claim 28, in a sample likely to contain in addition at least one toxinogenic cyanobacteria of the genus Aphanizomenon, comprising in addition to the optional hybridization step resulting from bringing said sample into contact with a capture probe and a signal probe specific to toxinogenic cyanobacteria of the genus Microcystis, an optional hybridization step resulting from bringing said sample into contact with a probe and a signal probe specific to toxinogenic cyanobacteria of the genus Aphanizomenon, the capture probe and the signal probe forming a pair of probes, the sequences of said pair of probes being selected from x elements of one of the following sets: (SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22) (SEQ ID NO: 23 and SEQ ID NO: 24) x being 2 or 3, or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, hybridization indicating the presence of toxinogenic cyanobacteria of the genus Aphanizomenon, preferably, the minimum detection threshold for the toxinogenic cyanobacteria of the genus Aphanizomenon being estimated from 10 to 575 active living cells per millilitre of sample (cells/mL), or less than or equal to 1.0 ng of ribosomal RNA per millilitre of sample, and in particular from 0.02 ng to 0.7 ng of ribosomal RNA per millilitre of sample and preferably from 0.02 ng to 0.1 ng of ribosomal RNA per millilitre of sample.

30. The method for the detection of toxinogenic cyanobacteria according to claim 28, in a sample likely to contain in addition at least one toxinogenic cyanobacteria of the genus Dolichospermum, comprising in addition to the optional hybridization step resulting from bringing said sample into contact with a capture probe and a signal probe specific to toxinogenic cyanobacteria of the genus Microcystis, An optional hybridization step resulting from bringing said sample into contact with a probe and a signal probe specific to toxinogenic cyanobacteria of the genus Dolichospermum, the capture probe and the signal probe forming a pair of probes, the sequences of said pair of probes being selected from x elements of one of the following sets: (SEQ ID NO 25 and SEQ ID NO 26) x being 2, or the sequences of said probes having at least 92% identity with the aforementioned sequences SEQ ID NO: 25, SEQ ID NO: 26, hybridization indicating the presence of toxinogenic cyanobacteria of the genus Dolichospermum, preferably, the minimum detection threshold for the toxinogenic cyanobacteria of the genus Dolichospermum being estimated from 10 to 575 active living cells per millilitre of sample (cells/mL), or less than or equal to 1.0 ng of ribosomal RNA per millilitre of sample, and in particular from 0.02 ng to 0.7 ng of ribosomal RNA per millilitre of sample and preferably from 0.02 ng to 0.1 ng of ribosomal RNA per millilitre of sample.

31. The method for the detection of toxinogenic cyanobacteria according to claim 28, in a sample likely to contain in addition at least one toxinogenic cyanobacteria of the genus Anabaena, comprising in addition to the optional hybridization step resulting from bringing said sample into contact with a capture probe and a signal probe specific to toxinogenic cyanobacteria of the genus Microcystis, an optional hybridization step resulting from bringing said sample into contact with a probe and a signal probe specific to toxinogenic cyanobacteria of the genus Anabaena, the capture probe and the signal probe forming a pair of probes, the sequences of said pair of probes being selected from x elements of one of the following sets: (SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29) (SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32) (SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35) (SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39) (SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42) (SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45) (SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48) (SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51) (SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54) (SEQ ID NO: 55 and SEQ ID NO: 56) (SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59) (SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62) x being 2, 3 or 4, or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, hybridization indicating the presence of toxinogenic cyanobacteria of the genus Anabaena, preferably, the minimum detection threshold of the toxinogenic cyanobacteria of the genus Anabaena being estimated from 10 to 575 active living cells per millilitre of sample (cells/mL), or less than or equal to 1.0 ng of ribosomal RNA per millilitre of sample, and in particular from 0.02 ng to 0.7 ng of ribosomal RNA per millilitre of sample and preferably from 0.02 ng to 0.1 ng of ribosomal RNA per millilitre of sample.

32. The method for the detection of toxinogenic cyanobacteria according to claim 28, in a sample likely to contain in addition at least one toxinogenic cyanobacteria of the genus Planktothrix, comprising in addition to the optional hybridization step resulting from bringing said sample into contact with a capture probe and a signal probe specific for toxinogenic cyanobacteria of the genus Microcystis, an optional hybridization step resulting from the contact of said sample with a probe and a signal probe specific to toxinogenic cyanobacteria of the genus Planktothrix, the capture probe and the signal probe forming a pair of probes, the sequences of said pair of probes being selected from x elements of one of the following sets: (SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66) (SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69) x being 3 or 4, or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, hybridization indicating the presence of toxinogenic cyanobacteria of the genus Planktothrix, preferably, the minimum detection threshold of the toxinogenic cyanobacteria of the genus Planktothrix being estimated from 10 to 575 active living cells per millilitre of sample (cells/mL), or less than or equal to 1.0 ng of ribosomal RNA per millilitre of sample, and in particular from 0.02 ng to 0.7 ng of ribosomal RNA per millilitre of sample and preferably from 0.02 ng to 0.1 ng of ribosomal RNA per millilitre of sample.

33. The method for the detection of toxinogenic cyanobacteria according to claim 28, wherein, said capture probe is linked to at least one attachment molecule positioned 5′ to its sequence and said signal probe is linked to at least one maring molecule positioned 5′ to its sequence, or said capture probe is linked to at least one attachment molecule positioned 5′ of its sequence and said signal probe is linked to at least one marking molecule positioned 3′ of its sequence, or said capture probe is linked to at least one attachment molecule positioned 3′ of its sequence and said signal probe is linked to at least one marking molecule positioned 5′ of its sequence, or said capture probe is linked to at least one attachment molecule positioned 3′ of its sequence and said signal probe is linked to at least one marking molecule positioned 3′ of its sequence, said at least one attachment molecule being in particular selected from a biotin, avidin, streptavidin, a thiol group, an amine group and a carbon, preferably a biotin molecule, the said at least one labelling molecule being chosen in particular from a fluorochrome, a biotin, a biotin-bound molecule, digoxigenin, an enzyme using a chemiluminescent substrate, an enzyme using a chromogenic substrate or an enzyme using an electrochemically oxidized substrate, preferably digoxigenin, preferably, said enzyme using a chromogenic substrate is alkaline phosphatase and said chromogenic substrate is Tetrazolium Nitroblue (NBT) or Bromochlorylindolophosphate (BCIP), or said enzyme using a chromogenic substrate is horseradish peroxidase (HRP) and said chromogenic substrate is selected from 3,3′-Diaminobenzidine (DAB), 3,3′,5,5′-Tetramethylbenzidine (TMB), or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS).

34. A pair of probes for the detection of toxinogenic cyanobacteria whose sequences are chosen from x elements of one of the following sets: (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4) (SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7) (SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11) (SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14) (SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17) (SEQ ID NO: 18 and SEQ ID NO: 19) x being 2, 3 or 4 or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, for the detection of toxinogenic cyanobacteria of the genus Microcystis, or pair of probes whose sequences are chosen from x elements of one of the following sets: (SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22) (SEQ ID NO: 23 and SEQ ID NO: 24) x being 2 or 3, or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, for the detection of toxinogenic cyanobacteria of the genus Aphanizomenon, or pair of probes whose sequences are chosen from x elements of one of the following sets: (SEQ ID NO: 25 and SEQ ID NO: 26) x being 2, or the sequences of said probes having at least 92% identity with the aforementioned sequences SEQ ID NO: 25, SEQ ID NO: 26, for the detection of toxinogenic cyanobacteria of the genus Dolichospermum, or pair of probes whose sequences are chosen from x elements of one of the following sets: (SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29) (SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32) (SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35) (SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39) (SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42) (SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45) (SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48) (SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51) (SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54) (SEQ ID NO: 55 and SEQ ID NO: 56) (SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59) (SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62) x being 2, 3 or 4, or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, for the detection of toxinogenic cyanobacteria of the genus Anabaena, or pair of probes whose sequences are chosen from x elements of one of the following sets: (SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66) (SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69) x being 3 or 4, or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, for the detection of toxinogenic cyanobacteria of the genus Planktothrix.

35. The pair of probes according to claim 28 for the detection of toxinogenic cyanobacteria, the sequences of the probes of the said pairs being as follows: (SEQ ID NO: 1 and SEQ ID NO: 2), (SEQ ID NO: 1 and SEQ ID NO: 3), (SEQ ID NO: 1 and SEQ ID NO: 4), (SEQ ID NO: 2 and SEQ ID NO: 3), (SEQ ID NO: 3 and SEQ ID NO: 4) (SEQ ID NO: 5 and SEQ ID NO: 6), (SEQ ID NO: 5 and SEQ ID NO: 7), (SEQ ID NO: 6 and SEQ ID NO: 7) (SEQ ID NO: 8 and SEQ ID NO: 9), (SEQ ID NO: 8 and SEQ ID NO: 10), (SEQ ID NO: 8 and SEQ ID NO: 11), (SEQ ID NO: 9 and SEQ ID NO: 10), (SEQ ID NO: 9 and SEQ ID NO: 11), (SEQ ID NO: 10 and SEQ ID NO: 11) (SEQ ID NO: 12 and SEQ ID NO: 13), (SEQ ID NO: 12 and SEQ ID NO: 14), (SEQ ID NO: 13 and SEQ ID NO: 14) (SEQ ID NO: 15 and SEQ ID NO: 16), (SEQ ID NO: 15 and SEQ ID NO: 17), (SEQ ID NO: 16 and SEQ ID NO: 17) (SEQ ID NO: 18 and SEQ ID NO: 19), for the detection of toxinogenic cyanobacteria of the genus Microcystis, or the sequences of the probes of the said pairs being as follows: (SEQ ID NO: 20 and SEQ ID NO: 21), (SEQ ID NO: 20 and SEQ ID NO: 22), (SEQ ID NO: 21 and SEQ ID NO: 21) (SEQ ID NO: 23 and SEQ ID NO: 24), for the detection of toxinogenic cyanobacteria of the genus Aphanizomenon, or the sequences of the probes of the said pairs being as follows: (SEQ ID NO: 27 and SEQ ID NO: 28), (SEQ ID NO: 27 and SEQ ID NO: 29), (SEQ ID NO: 28 and SEQ ID NO: 29) (SEQ ID NO: 30 and SEQ ID NO: 31), (SEQ ID NO: 30 and SEQ ID NO: 32), (SEQ ID NO: 31 and SEQ ID NO: 32) (SEQ ID NO: 33 and SEQ ID NO: 34), (SEQ ID NO: 33 and SEQ ID NO: 35), (SEQ ID NO: 34 and SEQ ID NO: 35) (SEQ ID NO: 36 and SEQ ID NO: 37), (SEQ ID NO: 36 and SEQ ID NO: 38), (SEQ ID NO: 36 and SEQ ID NO: 39), (SEQ ID NO: 37 and SEQ ID NO: 38), (SEQ ID NO: 37 and SEQ ID NO: 39), (SEQ ID NO: 38 and SEQ ID NO: 39) (SEQ ID NO: 40 and SEQ ID NO: 41), (SEQ ID NO: 40 and SEQ ID NO: 42), (SEQ ID NO: 41 and SEQ ID NO: 42) (SEQ ID NO: 43 and SEQ ID NO: 44), (SEQ ID NO: 43 and SEQ ID NO: 45), (SEQ ID NO: 44 and SEQ ID NO: 45) (SEQ ID NO: 46 and SEQ ID NO: 47), (SEQ ID NO: 46 and SEQ ID NO: 48), (SEQ ID NO: 47 and SEQ ID NO: 48) (SEQ ID NO: 49 and SEQ ID NO: 50), (SEQ ID NO: 49 and SEQ ID NO: 51), (SEQ ID NO: 50 and SEQ ID NO: 51) (SEQ ID NO: 52 and SEQ ID NO: 53), (SEQ ID NO: 52 and SEQ ID NO: 54), (SEQ ID NO: 53 and SEQ ID NO: 54) (SEQ ID NO: 55 and SEQ ID NO: 56) (SEQ ID NO: 57 and SEQ ID NO: 58), (SEQ ID NO: 57 and SEQ ID NO: 59), (SEQ ID NO: 58 and SEQ ID NO: 59) (SEQ ID NO: 60 and SEQ ID NO: 61), (SEQ ID NO: 60 and SEQ ID NO: 62), (SEQ ID NO: 61 and SEQ ID NO: 62), for the detection of toxinogenic cyanobacteria of the genus Anabaena, or the sequences of the probes of the said pairs being as follows: (SEQ ID NO: 63 and SEQ ID NO: 64), (SEQ ID NO: 63 and SEQ ID NO: 65), (SEQ ID NO: 63 and SEQ ID NO: 66), (SEQ ID NO: 64 and SEQ ID NO: 65), (SEQ ID NO: 64 and SEQ ID NO: 66), (SEQ ID NO: 65 and SEQ ID NO: 66) (SEQ ID NO: 67 and SEQ ID NO: 68), (SEQ ID NO: 67 and SEQ ID NO: 69), (SEQ ID NO: 68 and SEQ ID NO: 69), for the detection of toxinogenic cyanobacteria of the genus Planktothrix.

36. Kit for the detection of toxinogenic cyanobacteria of the genus Microcystis, said kit containing: a) at least one pair of probes specific to toxinogenic cyanobacteria of the genus Microcystis, the sequences of said probes being selected from x elements of one of the following sets: (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4) (SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7) (SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11) (SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14) (SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17) (SEQ ID NO: 18 and SEQ ID NO: 19) x being 2, 3 or 4 or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, one probe of said pair being a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ of its sequence and the other probe of said pair being a signal probe linked to at least one marking molecule positioned at 3′ or 5′ of its sequence, said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Microcystis, b) optionally a hybridization solution c) optionally a washing solution d) optionally one or more revelation solutions.

37. The kit according to claim 36, for the detection of toxinogenic cyanobacteria of the genus Microcystis and/or Aphanizomenon, said kit additionally containing: a) at least one pair of probes specific to toxinogenic cyanobacteria of the genus Aphanizomenon, the sequences of said probes being selected from x elements of one of the following sets: (SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22) (SEQ ID NO: 23 and SEQ ID NO: 24) x being 2 or 3, or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, one probe of said pair being a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ of its sequence and the other probe of said pair being a signal probe linked to at least one marking molecule positioned at 3′ or 5′ of its sequence, said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Aphanizomenon.

38. The kit according to claim 36, for the detection of toxinogenic cyanobacteria of the genus Microcystis and/or Dolichospermum, said kit additionally containing: a) at least one pair of probes specific to toxinogenic cyanobacteria of the genus Dolichospermum, the sequences of said probes being selected from x elements of one of the following sets: (SEQ ID NO: 25 and SEQ ID NO: 26) x being 2, or the sequences of said probes having at least 92% identity with the aforementioned sequences SEQ ID NO: 25, SEQ ID NO: 26, one probe of said pair being a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ of its sequence and the other probe of said pair being a signal probe linked to at least one marking molecule positioned at 3′ or 5′ of its sequence, said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Dolichospermum.

39. The kit according to claim 36, for the detection of toxinogenic cyanobacteria of the genus Microcystis and/or Anabaena, said kit additionally containing: a) at least one pair of probes specific to toxinogenic cyanobacteria of the genus Anabaena, the sequences of said probes being selected from x elements of one of the following sets: (SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29) (SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32) (SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35) (SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39) (SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42) (SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45) (SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48) (SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51) (SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54) (SEQ ID NO: 55 and SEQ ID NO: 56) (SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59) (SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62) x being 2, 3 or 4, or the sequences of said probes having at least 92% identity with the aforementioned sequences SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, one probe of said pair being a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ of its sequence and the other probe of said pair being a signal probe linked to at least one marking molecule positioned at 3′ or 5′ of its sequence, said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Anabaena.

40. The kit according to claim 36, for the detection of toxinogenic cyanobacteria of the genus Microcystis and/or Planktothrix, said kit additionally containing: a) at least one pair of probes specific to toxinogenic cyanobacteria of the genus Planktothrix, the sequences of said probes being selected from x elements of one of the following sets: (SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66) (SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69) x being 3 or 4, or the sequences of said probes having at least 92% identity with the abovementioned sequences SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, one probe of said pair being a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ of its sequence and the other probe of said pair being a signal probe linked to at least one marking molecule positioned at 3′ or 5′ of its sequence, said capture probe and said signal probe being capable of hybridizing with the ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Planktothrix.

41. The kit according to claim 36, said kit additionally including a support, said support being in particular selected from the group consisting of: a microplate, a glass slide, magnetic beads, electrodes printed in different materials such as carbon or gold, preferably a microplate or magnetic balls.

Description

[1325] The following figures and examples will better illustrate the invention, without limiting its scope.

[1326] FIG. 1: Average concentration of RNA content per cell, expressed in pg RNA/cell, obtained from optimal culture conditions corresponding to the exponential phase of growth of different toxinogenic cyanobacteria.

[1327] FIG. 2: Specificity test of probes targeting Anabaena and/or Aphanizomenon. Hybridization of 50 ng RNA extracted from an Anabaena culture, 10 nM positive control (PC, complementary synthetic DNA) or 10 nM negative control (NC, non-complementary synthetic DNA) with the following pairs of probes: SEQ ID NO 28 and SEQ ID NO: 29, SEQ ID NO: 33 and SEQ ID NO: 34 or SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 40 and SEQ ID NO: 41, SEQ ID NO: 46 and SEQ ID NO: 47.

[1328] FIG. 3: Correspondence between RNA concentration (pg/4) and absorbance at 450 nm: Nodularia (BCC1657 strain) and sequence probes SEQ ID NO: 76 and SEQ ID NO: 77 (A.), Microcystis (728.11 strain) and sequence probes SEQ ID NO: 1 and SEQ ID NO: 2 (B.); Microcystis (FL strain) and sequence probes SEQ ID NO: 1 and SEQ ID NO: 2 (C.).); Planktothrix (TCC strain 24) and sequence probes SEQ ID NO: 67 and SEQ ID NO: 68 (D.); Planktothrix (TCC strain 83.1) and sequence probes SEQ ID NO: 67 and SEQ ID NO: 68 (E.); Planktothrix (TCC strain 14) and sequence probes SEQ ID NO: 67 and SEQ ID NO: 68 (F.); Planktothrix (TCC strain 779) and sequence probes SEQ ID NO: 67 and SEQ ID NO: 68 (G.).

[1329] FIG. 4: Comparison of Microcystis detection by microscopy cell counts (cells/mL) and signals from the sandwich hybridization test according to the invention read at 450 nm (BIOCAPTER data). The results are reported per 1 mL of sample.

[1330] FIG. 5: Comparison of Planktothrix detection by microscopy cell counts (cells/mL) and signals from the sandwich hybridization test according to the invention read at 450 nm (BIOCAPTER data).

[1331] FIG. 6: Comparison of Planktothrix detection by microscopy cell counts (cells/mL) and signals from the sandwich hybridization test according to the invention read at 450 nm (BIOCAPTER data).

[1332] FIG. 7: Comparison of Planktothrix detection by microscopy cell counts (cells/mL) and signals from the sandwich hybridization test according to the invention read at 450 nm (BIOCAPTER data).

EXAMPLE 1: PROBE VALIDATION

[1333] a) Growing Conditions

[1334] The cyanobacterial cultures used to test the probes described in this invention are listed in Table 1. All the cultures are currently maintained at Microbia Environnement on the business incubation site of the Oceanological Observatory of Banyuls-sur-mer, France. The cultures are maintained in BG11 media proposed by MM Allen and RY Stanier (Selective isolation of blue-green algae from water and soil, J Gen Microbiol. 1968 April; 51(2): 203-9) at different temperatures (18 and 20° C.) and under a luminosity intensity of 100 pE 111.sup.−2 s.sup.−1 with a day: night cycle of 12:12. The BG11 medium is made from fresh or brackish water and is commonly used to cultivate cyanobacteria.

TABLE-US-00001 TABLE 1 Toxinogenic cyanobacteria used to test the probes described in the present invention, including class, growth medium and strain number Cultural Strain identification Genus CLASS environment number Anabaena Cyanophyceae BG11 TCC79 Aphanizomenon Cyanophyceae BG-11 PCC 7909 Microcystis Cyanophyceae BG-11 728.11 MNHN, FL Nodularia Cyanophyceae BG-11 1657, 1655 Planktothrix Cyanophyceae BG-11 TCC779, TCC83.1, Planktothrix Cyanophyceae BG-11 TCC14, TCC24

[1335] b) RNA Preparation

[1336] A known number of cells is filtered on a polycarbonate membrane with a porosity of 0.1 μm (Whatman® Nuclepore Track-Etched Membranes) using a filtration system and a vacuum pump. 1 ml TRI-Reagent (Sigma®, France) or 1 ml lysis buffer from the Quick-RNA™ MiniPrep kit (Zymo Research®, USA) is immediately added to each filtrate and homogenised. Cellular lysis is completed by adding beads (0.5 mm, Zymo Research®, USA) and applying vibration with a Tissue Lyser Mill (Qiagen®, USA) for 2 minutes at maximum speed.

[1337] Total RNAs are isolated using the Quick-RNA™ MiniPrep kit or by extraction with TriReagent. The RNA concentration is measured using a Nanodrop spectrophotometer (Peqlab®, Erlangen, Germany). The samples are either used immediately or stored at −80° C. until use.

[1338] The total RNAs of 10,000 to 500,000 cells were extracted in 3 replicates from different cultures and different strains of toxinogenic cyanobacteria. The RNA concentration values obtained were used to obtain an average value of RNA content per cell under optimal culture conditions (FIG. 1). Ayers et al (2005) assume that the exponential growth obtained under optimal culture growth conditions corresponds approximately to what happens during an efflorescence.

[1339] c) Design and Synthesis of Nucleic Probes

[1340] The probes of the present invention are synthesized according to the methods known to man of the art. They are rehydrated in ultrapure water to obtain a mother solution with a concentration of 100 μM. Oligonucleotide probes have been designated and tested for the toxinogenic cyanobacteria Anabaena, Aphanizonmenon, Nodularia, Microcystis and Planktothrix. (Table 2). The sequence probes SEQ ID NO: 28 and SEQ ID NO: 29 were used to test the Positive Control (PC) and Negative Control (NC).

TABLE-US-00002 TABLE 2 Oligonucleotide probes targeting Anabaena, Aphanizonienon Noduiaria, Microcystis and Planktothrix. Pair of probes tested (SEQ Sequences GC Sequences GC Species ID NO) (5′-3′) Tm (%) (5′-3′) Tm (%) PC (positif 28/29 GAC TCT TTA 60 43 CTG CGG ACC 60 61 control) ACA GCA GAC CTT TAC GCC ATA CAA TGC CAA TC (SEQ CAC (SEQ ID ID NO: 29) NO: 28) NC (negative 28/29 GAC TCT TTA 60 43 CTG CGG ACC 60 61 control ACA GCA GAC CTT TAC GCC ATA CAA TGC CAA TC (SEQ CAC (SEQ ID ID NO 29) NO: 28) Planktothrix 67/68 CTTACGGCAC 61 56 AGATTCCAGA 58 44 sp TCTCCCCTTTC GATGTCAAGT AAGG (SEQ ID CCTGGTA NO: 67) (SEQ ID NO: 68) Anabaena 28/29 GAC TCT TTA 60 43 CTG CGG ACC 60 61 sp ACA GCA GAC CTT TAC GCC ATA CAA TGC CAA TC (SEQ CAC (SEQ ID ID NO 29) NO: 28) Anabaena 33/34 CTC TGC CCC 63 58 GTAGTTT 54 40 sp GAC CAC ACT CCA CTG CTC CTA GCT TT TTA TTT GGT (SEQ ID (SEQ ID NO: 33) NO: 34) Aphanizomenon 20/21 AAT TCC CTC 61 58 CTA GCT TTG 56 42 sp TGC CCC GAC TAG TTT CCA CAC ACT CTG CTC TT (SEQ (SEQ ID ID NO: 20 NO: 21) Anabaena 40/41 GGC ACT TCC 57 41 ACC ACC TGT 59 54 sp ATC TTT CAA GTT CAC GTT YAG AAT TCG CCC GAA (SEQ (SEQ ID ID NO: 41) NO: 40) Anabaena 46/47 TTC ACG CTC 61 58 GAC GAC AGC 63 63 sp CCG AAG GCA CAT GCA CCA CTC CTA CCT GTG (SEQ (SEQ ID NO: 46) ID NO: 47) Microcystis  1/2 GCCAATTAGG 61 54 ATCGGGTATT 60 46 sp TTTCACCTBGC AGCAGTCGTT GGC AC TCCAACTG (SEQ ID (SEQ ID NO: 1) NO: 2 Nodularia 76/77 CTG AGC TAC 60 45 ACA TTG CTG 63 52 sp GGT TTT GTG TGT AGC TGC AGA TTT GCA CCT TTG TCC TC GT (SEQ ID (SEQ ID NO: NO: 76) 77)

[1341] d) Sandwich Hybridization Test

[1342] Probe specificity and sensitivity tests are carried out by sandwich hybridization. The biotinylated capture probe (SEQ ID NO: 28; SEQ ID NO: 33; SEQ ID NO: 20; SEQ ID NO: 40; SEQ ID NO: 46; SEQ ID NO: 76; SEQ ID NO: 1; SEQ ID NO: 67) is coupled to a neutravidin-functionalized solid support and a signal probe coupled to a digoxigenin molecule (SEQ ID NO: 29; SEQ ID NO: 34; SEQ ID NO: 21; SEQ ID NO: 41; SEQ ID NO: 47; SEQ ID NO: 77; SEQ ID NO: 2; SEQ ID NO: 68). The signal probe is placed in the presence of nucleic acid molecules that may contain the target ribosomal nucleic acid complementary to the capture and signal probes. The mixture is placed in the presence of the capture probe which will hybridize to its complementary targets forming a hybrid of three molecules: the capture probe, the signal probe and the target ribosomal nucleic acid. The hybrid complexes are revealed by the digoxigenin attached to the signal probe thanks to a colorimetric reaction initiated by a horseradish peroxidase-type enzyme with its substrate producing a blue colour. The intensity of the colour is proportional to the concentration of the target ribosomal nucleic acid. Using a calibration curve, the target nucleic acid concentration is associated with a number of toxinogenic cyanobacterial cells present in the sample being analysed.

[1343] The complete test is carried out in less than an hour.

[1344] The samples for the sandwich hybridization test are prepared as follows:

[1345] The culture cells are collected by filtration on a polycarbonate membrane (0.5 μm porosity; Whatman® Nuclepore Track-Etched Membranes). The membranes are transferred to a tube (Eppendorf®) containing 1 ml of TriReagent solution (Sigma®, France) and heated at 65° C. for 10 minutes. They are then subjected to the mill in the presence of 0.5 mm beads (Bashing Beads, Zymo Research®) for 1 minute at maximum speed. The supernatant is collected and 200 μL of chloroform is added and mixed. The samples are centrifuged for 15 minutes at 4° C. and the aqueous phase is transferred to a clean tube. 0.5 volume of isopropanol is added and the mixture is incubated for 1 hour at −20° C. After 20 minutes centrifugation at 9000 g at 4° C., the supernatant is removed and the pellet is washed twice with 70% ethanol. The pellets are dried in the open air and then solubilised in 50 to 100 μL of ultra pure water. The quantity and quality of RNA obtained are measured by spectrophotometry with NanoDrop (Thermo Scientific®) or NanoVue (Biochrom Spectrophotometers®). The total RNAs are fragmented using a solution comprising 40 mM Trizma base, pH 8.0/100 mM KOAc/30 mM MgOAc for 10 minutes at 65° C. before hybridization.

[1346] Hybridization steps are performed in a standard 96-well microplate (Nunc®, Denmark) functionalized with NeutrAvidin solution at 1 μg ml-1, incubated for 24 hours and washed with a saline solution such as PBS 1× (K.sub.2PO.sub.4, 0.1 M; KH.sub.2PO.sub.4, 0.1 M; KCl, 0.1 M, pH 7.6). The first hybridization step consists of mixing 200 ng RNA with the hybridization buffer (0.3 M NaCl, 0.08 M Tris-HCl, 0.04% SDS, pH 8) to a final volume of 100 μL containing the signal probe (1 mM), and heating at 60° C. for 10 minutes. The samples are then cooled and a final 0.05 M EDTA solution is added. The mixture is added to the microplate wells and incubated for 10 minutes at 60° C. The microplate is washed three times with a saline solution such as PBX 1×. 100 μL of anti-DIG-HRP antibody at a concentration of 75 mU/ml is then added and incubated for 15 minutes at room temperature. 100 μL of TMB is added and the absorbance is measured after 15 minutes of reaction at a wavelength of 630 nm. Then 50 μl of H.sub.2SO.sub.4 10% is added and the absorbance is immediately measured at a wavelength of 450 nm. Each step of the development is carried out at room temperature with constant agitation and shielded from light. Screening of the following pairs of probes: SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 33 and SEQ ID NO: 34 and SEQ ID NO: 20 and SEQ ID NO: 21 and SEQ ID NO: 40 and SEQ ID NO: 41 or SEQ ID NO: 46 and SEQ ID NO: 47 was conducted at the same hybridization temperature. The results were compared with those obtained on the positive (PC) and negative (NC) controls (FIG. 2). The positive control is a synthetic DNA fragment of 10 nM concentration complementary to both probes and the negative control is a synthetic DNA fragment of 10 nM concentration non-complementary to both probes. In general, the results obtained by sandwich hybridisation tests with samples from Anabaena cultures show good reactivity of the probes with the total RNA extracted. The SEQ ID NO: 20 and SEQ ID NO: 21 designated to recognise the genus Aphanizomenon cross-react with the Anabaena strain used. This result is explained by uncertainties in the current classification which is being revised as supported by Gugger et al. and Komarec who showed confusion in the classification of the genus Anabaena and Aphanizomenon (Gugger M., Lyra C., Henriksen P., Coute A., Humbert J-C. and Sivonen K. (2002). “Phylogenetic comparison of the cyanobacterial genera Anabaena and Aphanizomenon.” International Journal of Systematic and Evolutionary Microbiology, 52, 1867-1880; J Komarec (2010). “Modern taxonomic revision of planktic nostocacean cyanobacteria: a short review of genera.” Hydrobiologia 639:231-243).

[1347] For these reasons, the probes SEQ ID NO: 46 and SEQ ID NO: 47; and SEQ ID NO: 76 and SEQ ID NO: 77; and SEQ ID NO: 1 and SEQ ID NO: 2; and SEQ ID NO: 67 and SEQ ID NO: 68 have been chosen to produce the calibration curves.

[1348] e) Calibration Curves to Quantify Target RNAs

[1349] The best combinations of probes were used to establish the calibration curves. These calibration curves were developed from RNAs extracted from a determined number of algal cells from a culture as described in parts a) and b) of this example. A dilution of the RNAs was performed from 0.05 ng to 10 ng. Hybridization steps are performed as in part d) of this example. FIG. 3 shows the calibration curves obtained with the serial dilution at an absorbance of 450 nm for the following toxinogenic cyanobacteria: [1350] Nodularia (strain BCC 1657); probe pair tested: SEQ ID NO: 76 and SEQ ID NO: 77 (A.) [1351] Microcystis (strain 728.11); probe pair tested: SEQ ID NO: 1 and SEQ ID NO: 2 (B.) [1352] Microcystis (FL strain); probe pair tested: SEQ ID NO: 1 and SEQ ID NO: 2 (C.) [1353] Planktothrix (strain TCC 24); probe pair tested: SEQ ID NO: 67 and SEQ ID NO: 68 (D.) [1354] Planktothrix (strain TCC 83.1); probe pair tested: SEQ ID NO: 67 and SEQ ID NO: 68 (E.) [1355] Planktothrix (TCC 14 strain); probe pair tested: SEQ ID NO: 67 and SEQ ID NO: 68 (F.) [1356] Planktothrix (strain TCC 779); probe pair tested: SEQ ID NO: 67 and SEQ ID NO: 68 (G.).

[1357] The result shows a minimum detection threshold between 0.05 and 0.5 ng total RNA for each genus tested (FIG. 3).

EXAMPLE 2: DETECTION OF CYANOBACTERIA FROM NATURAL ENVIRONMENTAL SAMPLES

[1358] Implementation comparisons between the present invention and the traditional technique based on the identification and counting of cyanobacteria b microscopy with the Utermöhl method (1958) have been carried out on natural samples.

[1359] a) Detection of Microcystis on a Natural Body of Water

[1360] Microcystis monitoring was carried out on a basin located in Spain. Water samples were collected once a week on the surface. In parallel, a 50 ml sub-sample was collected, fixed to Lugol, and sedimented for 24 hours.

[1361] Microscopic counting according to the Utermöhl method, a reference method (Utermöhl VH, 1931. Neue wege in der quantitativen erfassung des planktons. Verh Int Verein Theor Angew Limnol 5: 567-595), required a 12-hour sedimentation stage followed by a careful counting of the cells under the microscope. The entire Utermöhl method was carried out in 24 to 48 hours. This method makes it possible to detect the presence or absence of toxinogenic cyanobacteria cells of the genus Microcystis and thus to determine the number of toxinogenic cyanobacteria cells of the genus Microcystis. However, this method cannot determine the activity of cells of toxinogenic cyanobacteria of the genus Microcystis.

[1362] The embodiment of the present invention was carried out in less than one hour and made it possible to determine the activity of cells of toxinogenic cyanobacteria of the genus Microcystis as well as the average number of living and active cells.

[1363] For each hybridization test, 10 millilitres of water were immediately filtered through polycarbonate membranes (porosity 0.1 μm; Whatman® Nuclepore Track-Etched Membranes). The membranes were transferred to a tube (Eppendorf®) containing 2 ml of ZR lysis solution (ZymoResearch®, USA) and heated at 65° C. for 10 minutes. They are then subjected to the mill in the presence of 0.5 mm beads (Bashing Beads, ZymoResearch®) for 1 minute at maximum speed. RNA extraction is performed with the QuickRNA® kit (ZymoResearch®, USA) with an elution volume of 180 μl of ultrapure water. The total RNAs are fragmented using a solution comprising 40 mM Trizma base, pH 8.0/100 mM KOAc/30 mM MgOAc) for 10 minutes at 65° C. prior to hybridization.

[1364] The hybridization steps are performed in a standard 96-well microplate (Nunc®, Denemark) functionalized with NeutrAvidin solution at 1 μg ml-1, incubated for 24 hours with the sequence probe SEQ ID NO: 1 at a concentration of 1 μM. After 24 hours the microplate is washed with a saline solution such as PBS 1× (K.sub.2PO.sub.4, 0.1 M; KH.sub.2PO.sub.4, 0.1 M; KCl, 0.1 M, pH 7.6). The RNA eluate is mixed with the hybridization buffer (0.3 M NaCl, 0.08 M Tris-HCl, 0.04% SDS, pH 8) to a final volume of 300 μl containing the sequence signal probe SEQ ID NO: 2 (1 mM). The hybridization mix is heated at 60° C. for 10 minutes, then a final 0.05 M EDTA solution is added. 100 μl of the mixture is dispensed into 3 wells of the microplate and incubated for 10 minutes at 60° C. The microplate is washed three times with a saline solution such as PBX 1×. 100 μL of anti-DIG-HRP antibody at a concentration of 75 mU/ml is then added and incubated for 15 minutes at room temperature. 100 μL of TMB are added and the absorbance is measured after 15 minutes of reaction at a wavelength of 630 nm. Then 50 μL of H.sub.2SO.sub.4 10% are added and the absorbance is immediately measured at a wavelength of 450 nm. Each step of the development is carried out at room temperature with constant agitation and shielded from light. In general, the results obtained by sandwich hybridization tests with naturally contaminated environmental samples are in agreement with the counts obtained by microscopy. However, the present invention makes it possible to obtain the results in less than one hour, compared with 24 to 48 hours for the counting method. The hybridization test has allowed the detection of Microcystis with the lowest microscopically determined concentration of 982 cells/mL in the environmental sample (FIG. 4). The known state of the art has never reported the detection of this type of cyanobacteria at such low cell equivalent thresholds in environmental samples. This threshold represents neither the limit of quantification nor the limit of detection in the environmental medium, which are well below what is shown by this environmental monitoring of Microcystis activity.

[1365] b) Detection of Planktothrix on a Natural Basin of Water

[1366] The monitoring of Planktothrix was carried out on a basin of water located in Spain from 23 Jul. to 7 Sep. 2018. Water samples were collected once a week on the surface. At the same time, a 50 ml sub-sample was collected, fixed to Lugol, and sedimented for 24 hours. Microscopic counting according to the Utermöhl method required a 12-hour sedimentation step followed by a careful counting of the cells under the microscope. The entire Utermöhl method was carried out in 24 to 48 hours. This method makes it possible to detect the presence or absence of cells of toxinogenic cyanobacteria of the genus Planktothrix and thus to determine the number of cells. However, this method cannot determine the activity of cells of toxinogenic cyanobacteria of the genus Planktothrix.

[1367] The embodiment of the present invention was carried out in less than one hour and made it possible to determine the activity of the cells of toxinogenic cyanobacteria of the genus Planktothrix as well as the number of cells.

[1368] For each hybridization test, 10 millilitres of water are immediately filtered through polycarbonate membranes (porosity 0.1 μm; Whatman® Nuclepore Track-Etched Membranes). The membranes are transferred to a tube (Eppendorf®) containing 2 ml ZR lysis solution (ZymoResearch®, USA) and heated at 60° C. for 10 minutes. They are then subjected to the mill in the presence of 0.5 mm beads (Bashing Beads, ZymoResearch®) for 2 minutes at maximum speed. RNA extraction is performed with the QuickRNA® kit (ZymoResearch®, USA) with an elution volume of 180 μl of ultrapure water. The total RNAs are fragmented using a solution comprising 40 mM Trizma base, pH 8.0/100 mM KOAc/30 mM MgOAc) for 10 minutes at 65° C. prior to hybridization and the samples are stabilized with a 0.05% EDTA solution.

[1369] The hybridization steps are performed in a standard 96-well microplate (Nunc®, Denemark) functionalized with NeutrAvidin solution at 1 μg/ml, incubated for 24 hours with the sequence probe SEQ ID NO: 67 at a concentration of 1 μM. After 24 hours the microplate is washed with a saline solution such as PBS 1× (K.sub.2PO.sub.4, 0.1 M; KH.sub.2PO.sub.4, 0.1 M; KCl, 0.1 M, pH 7.6). The eluate with RNA is mixed with the hybridization buffer (0.3 M NaCl, 0.08 M Tris-HCl, 0.04% SDS, pH 8) to a final volume of 300 μl containing the sequence probe SEQ ID NO: 68 at a concentration of 1 μM. The hybridization mix is heated at 60° C. for 10 minutes, then a final 0.05 M EDTA solution is added. 100 μl of the mixture is dispensed into 3 wells of the microplate and incubated for 15 minutes at 60° C. The microplate is washed three times with a saline solution such as PBS 1×. 100 μL of anti-DIG-HRP antibody at a concentration of 75 mU/ml is then added and incubated for 15 minutes at room temperature. 100 μL of TMB are added and the absorbance is measured after 15 minutes of reaction at a wavelength of 630 nm. Then 50 μL of 10% H.sub.2SO.sub.4 are added and the absorbance is immediately measured at a wavelength of 450 nm. Each step of the development is carried out at room temperature with constant agitation and shielded from light.

[1370] In general, the results obtained by sandwich hybridization tests with naturally contaminated environmental samples are in agreement with the counts obtained by microscopy. However, the present invention makes it possible to obtain the results in less than one hour, compared with 24 to 48 hours for the counting method. The hybridization test did not detect any Planktothrix cells as like the microscopy results which did not identify any Planktothrix (FIG. 5).

[1371] c) Detection of Aphanizomenon on a Natural Basin Water

[1372] The monitoring of Aphanizomenon was carried out on a basin of water located in France (Occitania) from 25 Jun. to 28 Oct. 2019. Water samples were collected twice a week on the surface. At the same time, a 50 ml sub-sample was collected, fixed to Lugol, and sedimented for 24 hours. Microscopic counting according to the Utermöhl method required a 12-hour sedimentation step followed by a careful counting of the cells under the microscope. The entire Utermöhlmethod was carried out in 24 to 48 hours. This method makes it possible to detect the presence or absence of cells of toxinogenic cyanobacteria of the genus Aphanizomenon and thus to determine the number of cells. However, this method cannot determine the activity of cells of toinogenic cyanobacteria of the genus Aphanizomenon.

[1373] The embodiment of the present invention was carried out in less than one hour and made it possible to determine the activity of the cells of toxinogenic cyanobacteria of the genus Aphanizomenon as well as the number of cells.

[1374] For each hybridization test, 250 millilitres of water are immediately filtered through polycarbonate membranes (porosity 0.1 μm; Whatman® Nuclepore Track-Etched Membranes). The membranes are transferred to a tube (Eppendorf®) containing 2 ml of ZR lysis solution (ZymoResearch®, USA) and heated at 60° C. for 10 minutes. They are then subjected to the mill in the presence of 0.5 mm beads (Bashing Beads, ZymoResearch®) for 2 minutes at maximum speed. RNA extraction is performed with the QuickRNA® kit (ZymoResearch®, USA) with an elution volume of 250 μl of ultrapure water. The total RNAs are fragmented using a solution comprising 40 mM Trizma base, pH 8.0/100 mM KOAc/30 mM MgOAc) for 10 minutes at 65° C. prior to hybridization and the samples are stabilized with a 0.05% EDTA solution.

[1375] The hybridization steps are performed in a standard 96-well microplate (Nunc®, Denemark) functionalized with NeutrAvidin solution at 1 μg/ml, incubated for 24 hours with the sequence probe SEQ ID NO: 20 at a concentration of 1 μM. After 24 hours the microplate is washed with a saline solution such as PBS 1× (K.sub.2PO.sub.4, 0.1 M; KH.sub.2PO.sub.4, 0.1 M; KCl, 0.1 M, pH 7.6). The eluate with RNA is mixed with the hybridization buffer (0.3 M NaCl, 0.08 M Tris-HCl, 0.04% SDS, pH 8) to a final volume of 300 μl containing the sequence probe SEQ ID NO: 68 at a concentration of 1 μM. The hybridization mix is heated at 60° C. for 10 minutes, then a final 0.05 M EDTA solution is added. 100 μl of the mixture is dispensed into 3 wells of the microplate and incubated for 15 minutes at 60° C. The microplate is washed three times with a saline solution such as PBS 1×. 100 μL of anti-DIG-HRP antibody at a concentration of 75 mU/ml is then added and incubated for 15 minutes at room temperature. 100 μL of TMB are added and the absorbance is measured after 15 minutes of reaction at a wavelength of 630 nm. Then 50 μL of H.sub.2SO.sub.4 10% are added and the absorbance is immediately measured at a wavelength of 450 nm. Each step of the development is carried out at room temperature with constant agitation and shielded from light.

[1376] In general, the results obtained by sandwich hybridization tests with naturally contaminated environmental samples are in agreement with the counts obtained by microscopy. However, the present invention makes it possible to obtain the results in less than one hour, compared with 24 to 48 hours for the counting method. The hybridization test has allowed the detection of Aphanizomenon, whose lowest concentration determined by microscopy is 969 cells/mL in the environmental sample (FIG. 6). The known state of the art has never reported the detection of this type of cyanobacteria at such low cell equivalent thresholds in environmental samples. This threshold represents neither the limit of quantification nor the limit of detection in the environmental medium, which are well below what is shown by this environmental monitoring of Aphanizomenon activity.

[1377] d) Detection of Planktothrix on a Basin of Water

[1378] The monitoring of Planktothrix was carried out on water basin located in France (Aquitaine) from 17 Jul. to 23 Oct. 2019. Water samples were collected in 2 sites once a week or once every 15 days on the surface. In parallel, a 50 ml sub-sample was collected, fixed to the Lugol, and sedimented for 24 hours. Microscopic counting according to the Utermöhlmethod required a 12-hour sedimentation step followed by a careful counting of the cells under the microscope. The entire Utermöhl method was carried out in 24 to 48 hours. This method makes it possible to detect the presence or absence of cells of toxinogenic cyanobacteria of the genus Planktothrix and thus to determine the number of cells. However, this method cannot determine the activity of cells of toxinogenic cyanobacteria of the genus Planktothrix.

[1379] The execution of the present invention was carried out in less than one hour and made it possible to determine the activity of the cells of toxinogenic cyanobacteria of the genus Planktothrix as well as the number of cells.

[1380] For each hybridization test and for each site, 50 millilitres of water are immediately filtered through polycarbonate membranes (porosity 0.1 μm; Whatman® Nuclepore Track-Etched Membranes). The membranes are transferred to a tube (Eppendorf®) containing 2 ml of ZR lysis solution (ZymoResearch®, USA) and heated at 60° C. for 10 minutes. They are then subjected to the mill in the presence of 0.5 mm beads (Bashing Beads, ZymoResearch®) for 2 minutes at maximum speed. RNA extraction is performed with the QuickRNA® kit (ZymoResearch®, USA) with an elution volume of 100 μl of ultrapure water. The total RNAs are fragmented using a solution comprising 40 mM Trizma base, pH 8.0/100 mM KOAc/30 mM MgOAc) for 10 minutes at 65° C. prior to hybridization and the samples are stabilized with a 0.05% EDTA solution.

[1381] The hybridization steps are performed in a standard 96-well microplate (Nunc®, Denemark) functionalized with NeutrAvidin solution at 1 μg/ml, incubated for 24 hours with the sequence probe SEQ ID NO: 67 at a concentration of 1 μM. After 24 hours the microplate is washed with a saline solution such as PBS 1× (K.sub.2PO.sub.4, 0.1 M; KH.sub.2PO.sub.4, 0.1 M; KCl, 0.1 M, pH 7.6). The eluate with RNA is mixed with the hybridization buffer (0.3 M NaCl, 0.08 M Tris-HCl, 0.04% SDS, pH 8) to a final volume of 300 μl containing the sequence probe SEQ ID NO: 68 at a concentration of 1 μM. The hybridization mix is heated at 60° C. for 10 minutes, then a final 0.05 M EDTA solution is added. 100 μl of the mixture is dispensed into 3 wells of the microplate and incubated for 15 minutes at 60° C. The microplate is washed three times with a saline solution such as PBS 1×. 100 μL of anti-DIG-HRP antibody at a concentration of 75 mU/ml is then added and incubated for 15 minutes at room temperature. 100 μL of TMB are added and the absorbance is measured after 15 minutes of reaction at a wavelength of 630 nm. Then 50 μL of 10% H.sub.2SO.sub.4 are added and the absorbance is immediately measured at a wavelength of 450 nm. Each step of the development is carried out at room temperature with constant agitation and shielded from light.

[1382] In general, the results obtained by sandwich hybridization tests with naturally contaminated environmental samples are in agreement with the counts obtained by microscopy. However, the present invention makes it possible to obtain the results in less than one hour, compared with 24 to 48 hours for the counting method. The hybridization assay allowed the detection of Planktothrix with the lowest microscopically determined concentration of 3083 cells/mL for the S2 site (FIGS. 7a) and 1116 cells/mL for the S6 site in the environmental samples (FIG. 7b). The known state of the art has never reported the detection of this type of cyanobacteria at such low cell equivalent thresholds in environmental samples. This threshold represents neither the limit of quantification nor the limit of detection in the environmental medium, which are well below what is shown by this environmental monitoring of Planktothrix activity.