Method for the specific detection of microorganisms
Inventors
Snaidr, Jiri • Beimfohr, Claudia • Muhlhahn, Peter
Assignees
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Publication Number
US-11680287-B2
Publication Date
2023-06-20
Expiration Date
Abstract
The invention relates to a method for the specific detection of a microorganism or a group of microorganisms via in situ hybridisation by means of flow cytometry.
Core Innovation
The invention relates to a method for detecting a microorganism in a sample using in situ hybridization in combination with a flow-through cytometer. Cells contained in the sample are fixed in place with a fixing agent and the fixed cells are separated from the sample. The fixed cells are brought into contact with a chemical homogenizing agent and dried to obtain dried cells, and then used for probe hybridization and detection.
The method performs sequential hybridization using a fluorescence marked nucleic acid probe specific for the microorganism and a quencher-marked nucleic acid probe comprising a quencher and a nucleic acid sequence substantially complementary to the nucleic acid sequence of the fluorescence marked nucleic acid probe. The fluorescence marked nucleic acid probe is incubated with the dried cells to bond to a corresponding target nucleic acid sequence in the cells of the microorganism. A second incubation is carried out with the quencher-marked nucleic acid probe to cause bonding of fluorescence marked nucleic acid probe molecules not bonded to the target sequence to the quencher-marked nucleic acid probe.
The second reaction mixture is then placed in a flow-through cytometer for detecting the fluorescence emitted from the cells containing the fluorescence marked nucleic acid probe. The chemical homogenizing agent comprises one of a monosaccharide and disaccharide, a polyol, and water. The document further supports specific probe and target choices, including ribosomal RNA, spacer sequences, transfer RNA, translation factor sequences, and messenger RNA sequences, as well as probe types and label arrangements described in dependent claims.
Claims Coverage
Two independent claims define substantially the same core method. Across the independent claims, the main inventive features are fixation and separation of cells, chemical homogenizing with a defined monosaccharide/disaccharide + polyol + water agent and drying, two-step hybridization with a fluorescence marked probe followed by a quencher-marked complementary probe, and fluorescence detection in a flow-through cytometer with quenching of unbound fluorescence.
Fixing, separating, and drying cells using chemical homogenizing
A method in which cells contained in the sample are fixed in place with a fixing agent and the fixed cells are separated from the sample; the fixed cells are brought into contact with a chemical homogenizing agent and dried, wherein the chemical homogenizing agent comprises one of a monosaccharide and disaccharide, a polyol, and water.
Sequential hybridization with fluorescence-marked nucleic acid probe and quencher-marked complementary probe
The dried cells are brought into contact with a fluorescence marked nucleic acid probe specific for the microorganism to bond to a corresponding target nucleic acid sequence; the first reaction mixture is then brought into contact with a quencher-marked nucleic acid probe comprising a quencher that at least partially quenches the fluorescence and a nucleic acid sequence substantially complementary to the fluorescence-marked probe, and incubated so that fluorescence-marked probe molecules not bonded to the target nucleic acid sequence bond to the quencher-marked nucleic acid probe.
Flow-through cytometer detection of fluorescence emitted from target-containing cells
The second reaction mixture is placed in a flow-through cytometer, and the fluorescence emitted from the cells of the microorganism containing the fluorescence-marked nucleic acid probe is detected.
The independent claims collectively cover a fixation, separation, chemical homogenizing, and drying workflow followed by sequential in situ hybridization using a fluorescence marked probe and a quencher-marked complementary probe, with detection performed in a flow-through cytometer.
Stated Advantages
Improved particle distribution and quantifiability in fluorescence detection.
Documented Applications
Detecting specific microorganisms in a sample using in situ hybridization with flow-through cytometry for direct qualitative and quantification of specific microorganisms.
Simultaneous detection of different microorganisms in a sample containing more than one microorganism.
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