The xTAG® Gastrointestinal Pathogen Panel (GPP) is a revolutionary multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of multiple viral, parasitic, and bacterial pathogens in human stool specimens from individuals with signs and symptoms of infectious colitis or gastroenteritis. As with other complex molecular tests, the package insert and good laboratory practices should always be followed, but here is a list of the top tips that will help minimize sample failures and/or reruns:
- Keep reagents COLD: The top tip for a successful GPP run is to keep reagents, including the master mix, cold while setting up PCR. Always work on a cold block and only remove the xTAG OneStep Enzyme Mix from the freezer when you are ready to use it. Return to the freezer immediately after use.
- Pre-heat thermal cyclers and heater block on Luminex analyzer: Preheat the thermal cyclers prior to the PCR (to 53°C) and bead hybridization step (to 60°C). The best way to preheat thermal cyclers is to program a hold step at the beginning of the cycling program. Preheat the XY platform on the Luminex analyzer (to 45°C). Preheating enhances specificity of the reactions.
- Controls: Positive and negative controls assure functionality of reagents and proper performance of the assay procedure. At least one negative and one positive control must be run with each batch of xTAG GPP run. We recommend running additional negative controls depending on the number of reactions. Controls should be prepared, extracted and tested in the same manner as patient samples. Different positive controls for each target can be used from run to run.
- Spin-Mix–Spin: Follow the “Spin-Mix-Spin” rule for all reagents when setting up the PCR reaction unless otherwise stated in the package insert. Spin to collect all of the reagents at the bottom of the tube, then vortex to efficiently mix and finally spin down to collect the reagents back at the bottom of the tube.
- xTAG OneStep Buffer: Once completely thawed, visually inspect the xTAG OneStep Buffer to ensure that there are no precipitates (including clear flakes) at the bottom. Vortex for 20 seconds to ensure all precipitates are dissolved. Do not centrifuge the OneStep Buffer.
- Unidirectional workflow: xTAG GPP assay should be run with a clearly defined workflow in specifically designated work areas. Assign separate areas for pre- and post-PCR activities. Fresh, clean gloves and lab coats must be worn in each area and must be changed before leaving that area. These precautions will reduce potential sources of contamination and ensure reliable results.
- Protect from light: xTAG GPP Bead Mix and the xTAG 0.22 SAPE (Note that SAPE from other Luminex assays are not interchangeable due to different SAPE formulations) are both sensitive to light. Avoid long exposures to direct light to prevent photo bleaching.
- Lot monitoring: As with all assays, do not use kits past expiry date or mix reagents from different kit lots. Expiration dates and lot numbers are both identified on the kit box.
- Environmental Monitoring: Establish a monthly environmental monitoring schedule to ensure effectiveness of cleaning routines. Monitoring should be performed using xTAG GPP with swab test samples collected from pre-PCR area surfaces such as centrifuges, pipettes, PCR racks, cold blocks, bench tops, refrigerators and freezer doors. It is important to remember not to swab post-PCR area as bringing samples from post-PCR area into pre-PCR area is not recommended.
- Pre-treatment step: Follow the guidelines for the recommended amount of stool to be used in the assay and perform the pre-treatment step as described in the package insert and in the Pre-treatment is Key in GPP blog.
For more information, view the product insert. If you are interested in learning more about our training options, visit Luminex Training
Products are region specific and may not be approved in some countries/regions. Please contact support@luminexcorp.com to obtain the appropriate information for your country of residence.