The rqmicro method
- The workflow is depicted in the following image:
- Concentration. The water sample is filtered to collect all bacteria on a polycarbonate filter. The filter is then transferred into a sample tube and the bacterial calls are resuspended in the rqmicro buffer.
- Incubation. rqmicro kit reagents are then added to the suspension. Antibodies bound to magnetic particles enable the isolation of target cells from the sample matrix by immunomagnetic separation (IMS). Antibodies bound to a fluorescent dye allow the quantification of target cells after IMS in a flow cytometer.
- Purification. The target cells, e.g. Legionella, are isolated and the background is reduced. This is done by an immunomagnetic separation, which is carried out either with the rqmicro.STREAM instrument in a microfluidic cartridge or with the Manual Immunomagnetic Separator (MIMS).
- Quantification. The purified target cells are then quantified using a flow cytometer. Alternatively, the sample can be further processed and be analyzed by qPCR, plating and culturing bacterial cells (standard method) or fluorescence microscopy.
- All aqueous solutions, e.g. various water matrices, blood, urine, saliva.
- Various food samples, e.g. milk, chocolate, meat
- Potable water
- Surface waters
- Cooling tower water
- Industrial water
- Environmental water
- Risk management and monitoring of water systems, e.g. drinking water, cooling water.
- Identification of Legionella hot spots in water systems.
- The specificity is guaranteed by the use of monoclonal antibodies, which bind only to the target cells, e.g, Legionella pneumophila.
- There are 2 different sets of monoclonal antibodies. The first set is coupled to magnetic particles and the second set is coupled to a green fluorochrome. Only if the target cell is recognized by both antibodies, the target cell is counted.
- The detection limit depends on the method of analysis, the water type and the assay, which is used. rqmicro recommends using a standard flow cytometer e.g. the Cytoflex (Beckman Coulter)
- Specifications are found in the data sheet of the assay.
- The viability dye, red, only stains dead, membrane-damaged, cells. Furthermore the antibodies which are coupled to a green fluorochrome stain all target cells present in a sample. As a result, the dead cells are stained red and green whereas the intact cells are only stained green. Distinguishing the intact cells from the dead cells is therefore easily done by flow cytometry.
The magnetic beads sediment in a matter of minutes.
- Bacterial cells remain undamaged during IMS and can be cultivated on agar after IMS.
How long can the positive fraction, purified and concentrated target cells in buffer, be stored before the analysis?
In order to obtain the best possible result, we recommend processing the positive fraction immediately after separation.
- rqmicro recommends the CytoFLEX (Beckman Coulter).
- With increasing complexity of the water matrix also the background is increasing. Depending on the assay, this can have an effect on the detection limit. Details are found in the data sheet of the assays.
- The flow cytometer needs to be equipped with an excitation laser at 488 nm and a green as well as a red fluorescence channels at 525/40 nm and 690/50 nm.