Air-Dryable Inhibitor-Tolerant qPCR Probe Mixes

Reliable amplification for various targets has been achieved using 40 cycles with 1 s denaturation and 3 s annealing and extension (2-step cycling). Successful amplification under these conditions requires reasonably short amplicons and should be tested on case-by-case basis for all new targets added to an experiment.

Results will differ depending on the plant species and tissue type. Our inhibitor-tolerant chemistry shows moderate resistance to chlorophyll and polyphenols, such as catechin and quercetin, but low resistance to tannic acid, cellulose and pectin. Thus, testing the mix on the desired sample types is recommend. All plant tissues will require mechanical lysis in an appropriate extraction buffer to achieve disruption of cell walls and membranes, prior to use with inhibitor-tolerant mixes. We recommend testing a small number of reactions on the target sample type. Please reach out to our team [email protected] to request a product sample, or if you need assistance in planning such test reactions.

Yes, this is expected. Crude biological samples, particularly saliva, contain different levels of PCR inhibitors. We have observed a range of 0.5-3.5 cycles delay in amplification of the same target with different saliva samples. The same crude biological sample may also show different levels of inhibition over time.

Yes. While the mix is inhibitor-tolerant, it is not completely resistant to the presence of inhibitors. Different inhibitors and higher concentrations can still inhibit qPCR reactions. Given the sensitivity of the mix, if a significant delay is observed, we recommend diluting the sample before using in qPCR reactions.

Saliva samples do not require pre-treatment. However, dilution in appropriate sample transport medium is recommended. We highly recommend the use of UTM which don’t contain guanidinum chloride.

We have successfully tested up to 10% crude saliva per reaction, with best, most-consistent results when using 2.5% saliva

We have successfully carried out multiplex reactions on up to 5 μL of blood sample (1.25 μL of crude blood diluted 1:4 in a NaCl/EDTA solution) per 20 μL reaction. This does lead to a reduction in overall fluorescence but does not inhibit qPCR reactions themselves, nor does it limit the sensitivity of the mix.

All our inhibitor-tolerant mixes have been tested on and tolerate crude saliva and crude blood. Additionally, they have been extensively tested in the presence of multiple PCR inhibitors found in clinical, plant, environmental, and laboratory chemicals . These include: hemin, haemoglobin, heparin, lactoferrin, IgG immunoglobulins, guanidine, SDS, ethanol, urea, sodium citrate, humic acid, cellulose, pectin, quercetin, catechin, tannic acid, and chlorophyll B. The extent of tolerance varies by compound. The mixes may also be tolerant to other compounds, not listed, or not yet tested.

The drying conditions provided in the product manual are only an example of the tested cycles used in our labs. When air-drying, it is important to get a loss of weight (Loss on Drying, LoD), of approximately 70-75 %, which results in a dried gel containing only 5-10 % water.

Drying times may vary depending on the instrument used. When using this product for the first time with a specific oven, we recommend measuring the LoD using multiple tubes at regular times (e.g., after 60, 70, 80, 90 min) to define the best protocol and achieve a LoD of 74.5 ± 10.5 % or 70 ± 1 % based on the mix (with or without oligos) and the drying instrument used.

Temperatures can also be changed, but obviously lower temperatures will require longer times, while higher temperatures risk affecting the integrity of the mix components, so we recommend using 40 °C to minimise the chances of unexpected problems.

We do not recommend drying more than 5 – 6 μL of mix (plus oligos) per well following the instructions provided into the manual. Multiple plates can be dried at the same time for high throughput of manufacturing. We do not recommend air-drying larger volumes and recommend lyophilisation, using our Lyo-Ready range of products.

Although we successfully managed to lyophilise this mix, we do not recommend it be lyophilised. The excipient content has been optimised for air drying and this significantly reduces stability of the product lyophilisation. We recommend using this mix for air-drying only and recommend our Lyo-Ready product range for lyophilisation protocols.