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The rqmicro method

  • The workflow is depicted in the following image:
  • In the first step, the bacterial cells are concentrated by filtration, which is also part of the standard method. The water sample is filtered with a standard filtration unit via a polycarbonate filter with a pore size of 0.22 μm, supplied in the rqmicro kits. The filter is then resuspended in a volume of 3 mL buffer.
  • Specific antibodies bound to magnetic particles as well as specific antibodies bound to a fluorescent dye are then added to the sample. The antibodies bound to the magnetic particles enable isolation of the target cells from the rest of the sample by means of the so-called immunomagnetic separation (IMS). The antibodies bound to a fluorescent dye allow the quantification of the bacterial cells after the immunomagnetic separation in a flow cytometer.
  • The sample is then incubated for 30 minutes at room temperature so that the antibodies can bind to the target cells.
  • During the separation process, which takes place either in the rqmicro CellStream instrument in a microfluidic cartridge or in the Manual Immunomagnetic Separator (MIMS), the bacterial target cells, for example, Legionella, are collected in a volume of 1 mL of clean buffer (second concentration step). With the rqmicro sample preparation a sample purity of more than 95% is achieved.
  • The positive fraction can then be measured and quantified using a commercially available flow cytometer. However, the sample can also be analyzed by PCR, by plating and culturing of bacterial cells (standard method), fluorescence microscopy, etc.
  • Antibodies are binding proteins that recognize and bind to a specific epitope (molecular structure, antigen) on the surface of a cell. The antibodies developed by rqmicro were selected to detect specific epitopes on the surface of e.g. Legionella.
  • B-cells of the immune system produce and secrete antibodies. Each B-cell (group of B cells) produces a specific antibody. Each antibody has a specific, characteristic antigen binding site.
  • Drinking water, surface waters, cooling tower water, sewage, industrial water, environmental water
  • rqmicro is working to ensure that also food samples can be analyzed with the rqmicro method in due course. The sample preparation must be adapted.
  • The test is particularly interesting for rapid testing in case of an outbreak in order to indentify the source as fast as possible –  but also for monitoring the progress during disinfection or heat treatments.
  • The sample preparation is highly specific. The specificity is guaranteed by the use of monoclonal antibodies, which bind only to the target cells, e.g, Legionella pneumophila.
  • The rqmicro antibody development team works on the discovery of proprietary antibodies which are coupled to magnetic particles –  they can then be used for performing immunomagnetic separation.
  • The detection limit depends on the method of analysis. rqmicro recommends using a standard flow cytometer e.g. the Attune (Thermo Fisher Scientific), Cytoflex (Beckman Coulter) or Accuri (Becton Dickinson)
  • Lower detection limit: approx. 20 – 50 bacterial cells
  • Higher detection limit in tap water: between 10^8 and 10^9 bacterial cells. If cell counts are extremely high, an option is to filter less sample.
  • If two distinct populations – the viable and the dead population – need to be identified in the same dot plot, the sample can be stained with two different fluorescent dyes.
  • The viability dye, red, only stains dead cells. The staining dye, green, stains all target cells present in a sample. As a result, the dead cells are stained red and green whereas the viable cells are only stained green. Like this, the viable and the dead cell population can easily be distinguished when analyzed with the flow cytometer.

Dot plots are one way of displaying the result of a flow cytometric analysis. In the plot on the left, all Legionella cells are stained green with the staining dye. By gating the Legionella population, the total Legionella cell count can be determined. In the plot on the right, the red viability stain was added to the sample, which only stains dead cells. Consequently, dead Legionella cells are stained green and red and shift upwards in contrast to the plot on the left.

Bacterial cells remain undamaged during IMS and can be cultivated on agar after IMS.

After the rqmicro sample preparation with the rqmicro kit and CellStream instrument the positive fraction can be analyzed by PCR, fluorescence microscopy, the cultivation of cells on agar, etc.

In order to obtain the best possible result, we recommend that the positive fraction be processed immediately after separation. The storage conditions of the processed positive fraction depend on the further intended use. If the sample is used for plating on agar it can be stored at room temperature for a couple of hours, otherwise we recommend storage of samples in the fridge.

During incubation and also during the CellStream process samples can be filtered and resuspended.

  • The workflow is significantly shorter (time savings)
  • It is possible to differentiate between viable and dead cells
  • Enrichment is unnecessary
  • Risk of false-negative results is significantly lower
  • The kit which is commercially available at the moment is only for the detection of L.p. SG1
  • L.p. SG1-15 und L. spp. will be available in the course of 2018.
  • rqmicro is working on the development and commercialization of further specific antibodies and tests. Kits under development include the detection of E. coli, Salmonella, Pseudomonas and Giardia and Cryptosporidium.
  • The rqmicro method has been validated internally. Customers have been benefiting from the internal laboratory service for 3 years.
  • AOAC and ISO certification are in planning, as well as round robin test to verify the correlation to the ISO method in different matrices.
  • rqmicro validated the kit for Legionella pneumophila SG1 internally. As soon as other kits are available, rqmicro is also going to validate them internally which will be followed by round robin tests.

Legionella detection with the rqmicro method

  • Filtration: 5 – 30 min / sample (depends on the volume that needs to be filtered and on the degree of pollution of the sample)
  • Handling: 5 min / sample (vortexing, transferring, adding the reagents)
  • Incubation: 30 min
  • Separation: 35 min.; the CellStream can process 4 samples in parallel
  • Detection: ca. 1 min / sample (with flow cytometry)
  • 60 – 90 min, 120 min maximum
Component Description Amount Reactions
Filter Polycarbonate filter; pore size 0.22 µm; Ø 47 mm 100 96
Buffer 1 Washing buffer 300 mL 96
Buffer 2 Separation buffer 300 mL 96
Magnetic particles Monoclonal antibody conjugated to magnetic beads 3 mL 96
Staining dye Labelling antibody for Legionella pneumophila SG1 cells; Ex/Em 493/518 nm 1 tube* 96
Viability dye Propidium iodide; penetrates membrane of dead cells and stains DNA; Ex/Em 535/617 nm 1 mL 96
Positive control Lyophilised, chemically fixed, freeze-dried Legionella pneumophila Philadelphia cells 1 tube 10

*Centrifuge for 2 min at maximum speed before use.

  • 25 microfluidic cartridges (96 reactions)
  • The results correlate (see brochure) with a 5-10 times higher sensitivity of the rqmicro method (IMS with subsequent flow cytometic analyisis).
  • One reason for this significantly higher sensitivy is, that the standard method only detects cells that do grow on agar plates. This is, in the case of Legionella, usually only a fraction of around 5% to 10% of the cells that are present in a sample. Flow cytometry however is a culture-independent method that detects all cells present in a sample. The rqmicro method consequently also detects VBNC (viable but non-culturable) bacteria. These bacteria can be infectious and if they are temporarily non-infectious, they are able to recover their infectivity under certain conditions. As they do not grow on agar plates, they are aslo not detected by the standard method.
  • Another reason for the higher sensitivity is that the CellStream process (IMS) removes 95% of non-target organisms initially present in a sample (sample purification). This renders the acid and/or heat treatments, which are usually done in order to eliminate the competing flora, obsolete. Since acid and/or heat treatments also harm Legionella and reduces their ability to grow on agar, false negative or markedly too low results often occur when performing the standard method. Bacterial cells isolated and purified with the rqmicro method are unharmed and grow well on agar plates.
  • The relative lack of competing flora is another advantage of the rqmicro method as high cell numbers of competing flora suppress the growth of Legionella.

In order to obtain the best possible result when resuspending the filter in buffer, rqmicro uses polished polycarbonate filters – the filtered cell material dissolves better from the surface of the filter.

The CellStream process (IMS) removes 95% of non-target organisms initially present in a sample (sample purification). This renders the acid and/or heat treatments, which are usually done in order to eliminate the competing flora, obsolete. Since acid and/or heat treatments also harm Legionella and reduces their ability to grow on agar, false negative or markedly too low results often occur when performing the standard method. Bacterial cells isolated and purified with the rqmicro method are unharmed and grow well on agar plates. The relative lack of competing flora is another advantage of the rqmicro method as high cell numbers of competing flora suppress the growth of Legionella.

The CellStream Instrument

  • During the separation process, which takes place in the CellStream instrument in a microfluidic cartridge, the sample is transported through a narrow channel from left to right. A vacuum pump adds positive or negative pressure in the corresponding containers of the cartridge and thereby creates this flux. During this process, a magnet is lowered down onto the microfluidic channels and thereby prevents the bacterial cells, which are bound to magnetic particles via specific antibodies, from continuing to flow in the channel (immobilization). Thus, the target cells are immobilized and at the same time > 95% of the non-target cells (competing flora) originally present in the sample reach the residual fraction (waste). This is followed by a wash step with buffer, which contributes to the high sample purity.
  • In the last step of the separation, the magnet is raised and as a result, the concentrated and purified target cells are eluted in 1 mL of clean buffer into the container of the positive fraction.

The immunomagnetic separation (IMS) takes place on a microfluidic cartridge in the CellStream instrument.

  • Approximately 44-48 samples can be processed in one working day (7 h) by one person; 12‘240 samples/year.

Temperature control in the CellStream device is not required. The components installed in the device are not heated during sample preparation.

We recommend annual maintenance at rqmicro or its distribution partners.

The following components of the device are included in the contract:

  1. Casing
  2. Mechanics
  3. Actuation/pneumatics
  4. Electronics including screen and connections

Scope of service:

  1. Two preventive maintenances of the device
  2. Unlimited number of repairs if required

 

The black rubber seal on the ceiling of the cartridge chamber should be cleaned before the first use and then approximately once a week. Use a mixture of 70% ethanol in water.

The positive fraction should be removed from the cartridge as soon as possible after the end of the separation process in order to avoid backflow. In addition, at the end of the process, air bubbles form in the channel going to positive fraction, which reduces the risk of reflux.

Flow Cytometry

Since the CellStream process removes 95% of the non-target organisms (competing flora) initially present in a sample, the positive fraction is clean and the background noise therefore low. The higher the initial cell number of the competing flora is in a specific sample, the higher is the amount of non-target cells that end up in the positive fraction. A higher background noise in comparison to drinking water is typical for complex matrix samples e.g. cooling tower samples.

rqmicro recommends the Attune (Thermo Fisher Scientific), the Cytoflex (Beckman Coulter) or the Accuri (Becton Dickinson).

  • The sample matrix does influence the result obtained by flow cytometry in that it influences the state of the bacterial cells: If there are many nutrients present in a sample matrix the cells may be larger (it also depends on temperature, competition, etc.) and their metabolic activity is higher when the cells are growing and dividing. The Life Scientist specialists at rqmicro gladly help how to interpret the FCM dot plots correctly.
  • During the immunomagnetic separation (IMS) in the CellStream, the bacterial target cells are separated from the rest of the sample and collected in a clean buffer (volume 1 mL). 95% of the non-target cells originally present in the sample (competing or background flora) are directed to the residual fraction (waste). In drinking water usually around 60 to 80 mio. cells are present in 1 L whereas e.g. in cooling tower water the cell count is usually a lot higher. The higher the initial cell number of the competing flora is in a specific sample, the higher is the amount of non-target cells that end up in the positive fraction. A higher background noise in comparison to drinking water is typical for complex matrix samples e.g. from cooling towers.
  1. Green fluorescence: Ex/Em 493/518 nm
  2. Red fluorescence: Ex/Em 535/617 nm
  3. Setting individual gates

If you didn’t find an answer to your question, please don’t hesitate to contact us.