Optimisation and Validation of PCR Method for HLA Gene Expression to Enable PCR System Transfer and Master Mix Change
Sammanfattning: Health Tech company Dynamic Code AB provides a PCR test for determination of HLA DQ-genes connected to development of celiac disease. The PCR method is probe based and in real time and is at this time carried through on the, somewhat outdated, PCR instrument from Thermo Fisher/Applied Biosystems called 7300 Real-Time PCR System. The run time for this analysis on the instrument is 1 hour and 50 minutes. The Master Mix in use is TaqMan™ Gene Expression Master Mix, from the same manufacturer. Moving on to a more modern PCR instrument is a natural step for the company and is favourable in several regards, one of them being the run time that will be cut by 50 minutes, allowing for more samples to be analysed in the same amount of time. The objective is to move the HLA analysis to Thermo Fisher’s QuantStudio™ 6 and 7 Flex Systems and at the same time change the Master Mix to SolisFast® Probe qPCR Mix (Purple) from Solis BioDyne, in order to achieve better accuracy as this Master Mix is more compatible with the latter instrument, along with reducing reagent cost as it is less expensive. In order to find the optimal primer and probe concentration for each target included in the HLA analysis, their concentrations were varied and tested with the new Master Mix on the new instrument. PCR instrument transfer and Master Mix change was successful and validation experiments showed a 98,9% accuracy for the new method compared to the original method.
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