Use of probes to detect toxinogenic cyanobacteria, detection method and corresponding kits
Inventors
GUILLEBAULT, Delphine • VILLA, Elisa • Crawford, Linda
Assignees
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Publication Number
US-12509735-B2
Publication Date
2025-12-30
Expiration Date
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.
Core Innovation
The invention relates to a sandwich hybridization technique for the detection of at least one toxinogenic cyanobacteria in a sample. The assay uses a capture probe and a signal probe specific to toxinogenic cyanobacteria of a target genus, and the presence of a hybridization complex indicates the presence of toxinogenic cyanobacteria in the sample.
The capture probe and the signal probe are capable of hybridizing with ribosomal nucleic acid, and the complex formed by this hybridization is detected. The capture probe is linked to at least one attachment molecule positioned at a 3′ or 5′ end of its sequence, and the signal probe is linked to at least one marking molecule positioned at a 3′ or 5′ end of its sequence.
Probe sequences and probe sequence sets are defined for multiple toxinogenic cyanobacteria genera, including Microcystis, Anabaena, Dolichospermum, Aphanizomenon, Nodularia, Planktothrix, and Cylindrospermopsis. The described embodiments use ribosomal RNA targeting to preferentially detect active/living cells, with attachment/marking systems using biotin/avidin/streptavidin and marking molecules such as digoxigenin, fluorochromes, and enzyme/chemiluminescent/chromogenic/electrochemical readouts.
Claims Coverage
The independent claims cover a Microcystis detection method, a Microcystis capture/signal probe pair, and a Microcystis detection kit. The common inventive framework is a capture probe and a signal probe linked to an attachment molecule and a marking molecule at defined 3′/5′ positions, hybridization to ribosomal nucleic acid, and detection based on the presence of the hybridization complex.
Microcystis sandwich hybridization detection with ribosomal nucleic acid hybridization complex
A method for the detection of at least one toxinogenic cyanobacteria of the genus Microcystis in a sample by contacting the sample with a capture probe and a signal probe specific to toxinogenic cyanobacteria of the genus Microcystis forming a pair of probes with defined SEQ ID NO pairings, wherein the capture probe is linked to at least one attachment molecule positioned at 3′ or 5′ end of its sequence and the signal probe is linked to at least one marking molecule positioned at 3′ or 5′ end of its sequence, the capture probe and the signal probe being capable of hybridizing with a ribosomal nucleic acid to form a complex, and detecting said hybridization complex, wherein presence of said hybridization complex indicates the presence of toxinogenic cyanobacteria of the genus Microcystis in the sample.
Microcystis capture and signal probe pair with defined end-linked attachment and marking molecules
A pair of probes for the detection of toxinogenic cyanobacteria of the genus Microcystis having sequences as SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 18 and SEQ ID NO: 19, wherein one probe is a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ end of its sequence and the other probe is a signal probe linked at least one marking molecule positioned at 3′ or 5′ end of its sequence.
Microcystis detection kit with capture/signal probe pairs and optional assay solutions
A kit for the detection of toxinogenic cyanobacteria of the genus Microcystis containing at least one pair of probes specific to toxinogenic cyanobacteria of the genus Microcystis with defined SEQ ID NO pairs, wherein one probe of said pair is a capture probe linked to at least one attachment molecule positioned at 3′ or 5′ end of its sequence and the other probe is a signal probe linked to at least one marking molecule positioned at 3′ or 5′ end of its sequence, said capture probe and said signal probe being capable of hybridizing with a ribosomal nucleic acid of a toxinogenic cyanobacteria of the genus Microcystis, and optionally a hybridization solution, optionally a washing solution, and optionally one or more revelation solutions.
The core coverage centers on sandwich hybridization using end-positioned attachment and marking molecules on capture and signal probes with defined SEQ ID NO pairings, hybridization to ribosomal nucleic acid, and detection where presence of the hybridization complex indicates presence of toxinogenic cyanobacteria of the genus Microcystis.
Stated Advantages
Provides a rapid assay time compared with microscopy.
Revealed activity determination aligned with microscopically determined concentration thresholds in natural environmental samples.
Very low limits of detection, reported as 0.02–0.7 ng ribosomal RNA.
Under 1 hour detection time for quantifying ribosomal nucleic acids from living toxinogenic cyanobacteria.
Reported correlation with active/living cells, reported as 10–575 active cells/mL.
Preferential detection of active/living cells by targeting ribosomal RNA.
Faster detection than Utermöhl microscopy is explicitly noted.
Documented Applications
Detection of toxinogenic cyanobacteria in natural environmental samples, including environmental sample testing with results aligned with microscopically determined concentration thresholds.
Assay validation and detection for Planktothrix targeting, using Planktothrix-targeting probe sequences and sandwich hybridization to detect target ribosomal nucleic acid.
Detection and quantification of ribosomal nucleic acids from living toxinogenic cyanobacteria using sandwich hybridization.
Detection of toxinogenic cyanobacteria in a sample for the genus Microcystis and, in dependent embodiments described in the partial content, additional genera including Aphanizomenon, Dolichospermum, Anabaena, Planktothrix, Nodularia, and Cylindrospermopsis.
Use of a kit containing probe pairs with optional hybridization solution, washing solution, and one or more revelation solutions for detection of toxinogenic cyanobacteria.
Performance assessment and comparison using Utermöhl microscopy.
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