Methods for Testing Pathogens

Lateral flow sheets are easier to use and require little equipment. A sample after enrichment is placed onto the sample pad. The sample diluent in the sample flow across through capillary action past the conjugate pad. Here the antigen i.e. the bacteria picks up a colloidal gold conjugate and carries it towards the test line where antibody bound onto nitrocellulose strip will pick up the antigen bound conjugate. The conjugate is colored and show up as a colored line. Conjugate that do not pick up any antigen will be carried past the test line to the control line where it will show up as another colored line. Positive samples will have two colored lines and negative one line at the control position.

 

Enzyme substrate technology uses a reagent which has unique enzyme substrates targeting the specific bacteria like campylobacter or E. coli/coliform. The enzyme and the substrate when bound together does not fluorescence but when the specific enzyme is present, meaning that the specific bacteria is present and thriving, the substrate will be consumed. This leaves the enzyme to be free and with a chromogen present will fluorescence under UV light of the appropriate wavelength. 1ml of sample is added to a sterile test tube together with 9ml of water. To activate the growth of bacteria, a growth nutrient in dehydrated form, supplied with the kit, is added to the test tube. The solution is mixed well and the contents poured onto the SimPlate provided. This plate has multiple dimples or wells which will have enough nutrients for 1cfu (for each well). The dimples are separate from each other and this prevents spreading and makes counting of discrete colonies easier. The cover is then laid on top of the plate and incubated for a specified time. Wells that show a color change and or fluorescence are counted and enumeration is by referring to a MPN table.

The enzyme substrate technology in 100ml bottles also uses enzyme substrate technology like the above method. Because enumeration or detection of pathogens in water requires a sample of 100ml according to the standard method for water and waste water, a sample bottle of 100ml is provided. After mixing the sample and nutrient substrate in the bottle supplied, the bottle is incubated for an absence or presence determination. Results are as shown in the above illustration. For quantification the contents of the bottle is emptied into a tray called Quanti-Tray, sealed with a sealing machine and incubated. Quatification is by counting the color change and/or fluorescence in the wells and comparing it to a MPN table.

The PCR technology detects pathogen by comparing the DNA of the investigated bacteria with the DNA of the conserved regions of the target. The sample is first enriched with specific broths to grow the target organism to detectable levels. To reduce the interference of matrix in food samples, the pathogen is separated from the mixture using immuno magnetic separation. For easy and quick separation BioControl uses a multiple magnetic pipette tips called a Pick Pen. The target pathogen is then re-suspended in a buffer and transferred to an amplification tube with specific probes inside which will identify the specific DNA regions and amplify them. The melting curves for each cycle of the thermo cycler, called rotor gene is, stored and analyzed in the instrument software.