1) Sample preparation. Sample preparation and homogenization (as necessary) of the item sampled for test. This step is heavily dependent on the matrix (food item or food process area swab; liquid or solid food matrix; high/low matrix complexity, animal product or vegetable) and deploys existing standard sample identification and preparation protocols for subsequent accredited tests for food pathogens.
Regulatory SOPs have been laid out , commercial solutions for large-scale sample preparation for standard microbiology and PCR also are available . In a large-scale routine laboratory setting, VPCIR may thus be adapted to fit into the individual laboratory’s pre-existing sample preparation and data management system, whether this had been based on culture or PCR.
Importantly, there is no pre-cultivation step involved as VPCIR benefits from an extreme high sensitivity. The outcome of Step 1 is 1 ml homogenized samples in 2 ml tubes, ready for subsequent testing, duly identified in the laboratory’s data management system.
2) Encapsulation of single cells into droplets and substrate conversion reaction. The homogenized samples are each mixed with VPCIR reagents, hydrogel beads functionalized with specific DNA substrates, and bacteria-specific bacteriophages in the wells of standard 96 wells plate by an automated robot . Through a rapid pipetting mix, bacteria cells are encapsulated into droplets together with the VPCIR reagents, beads and bacteriophages.
The time duration of this step is 5 minutes. While kept at room temperature, the VPCIR reactions takes place. Inside the droplets, the bacteria are lysed by the phages and the specific restriction enzymes are released converting the DNA substrates which are coupled to the beads.
The converted substrates are isothermally (at room temperature) amplified by Rolling Circle Amplification together with fluorescent molecular beacons giving rise to fluorescent beads. The time for these steps is 1 h and 30 min. The outcome of Step 3 is (duly identified) samples in a 96-well plate, now ready for readout.
3) Read-out. The result is read on a high throughput flow cytometer . In case of positive result, colors (in the case of Listeria at three different wavelengths) will be displayed. The total time to read-out result is 10 minutes. The outcome of Step 3 is a test result (duly identified) that are transferred to the laboratory’s data management system.
Using the VPCIR for testing food items for pathogen contamination is fast and easy to perform. Moreover, for an established analytic laboratory there will be no additional costs, as all the consumables are provided in the VPCIR kit, whereas standard off-the-shelf materials and equipment may be deployed that most laboratories are equipped with, rendering VPCIR with a plug-and-play capability.