Pseudomonas aeruginosa - FAQ
Is the Real time PCR test (mex Q-TesT) still valid even if the primers mix and the calibration standards arrive defrosted?
No, the mex Q-TesT kit (including primer-Mix and calibration standards) must be kept at -20 °C during the time of transport. We guarantee the stability of these reagents if the cold chain at -20°C has not been broken.
How many cycles of freeze / thaw of -20 ° C can calibration standards undergo without reducing the accuracy of quantification of the test?
The calibration standards allow 10 cycles of freeze / thaw without any negative effects on their effectiveness.
Is the amount of 1 µg of RNA used for RT step must be so very accurate?
No, minor differences are acceptable because there will be a normalization of signals by two housekeeping genes from the RT-PCR.
What happens if the OD of the bacterial culture is out of 1 +/- 0.1?
It is not recommended. Any excess OD of cultures induces false-positive in the final analysis, primarily with the mexA target.
Should we check the efficiency of DNase used after RNA extraction step?
It is recommended. Efficiency of DNase can be check with HKG1 and HKG2 amplification. Purified RNA can be directly used as template in PCR with HKG1 and HKG2 ampli-mix. No amplification indicates that the RNA is properly purified without DNA contaminant. This indirectly proves the efficiency of the DNase enzyme.
Can we use the wild-type strain BAA-47 from the ATCC list as a negative control?
We do not advise using this strain. This strain has an expression of mexX too high suggesting a resistance to antibiotics.
Which positive control should be used to validate the conditions for using the kit?
None. The kit provides calibration standards for the four genes identified (mexA, mexX, HKG1, HKG2 genes), which act as positive control.
Does the wild type PAO1 strain is supplied?
No, the strain must be acquired by the user in reference laboratories