we have a problem in detecting SARS-COV2 samples where the same samples show different results positive and negative when using different extraction kits on different instruments . so how we can rely on the cutoffs provided by the commercial kits which most of them have calculated their cutoffs ,without applying a standard calibration curve as in our case which result should we accept ?
Each PCR kit recommend specific extarction kit and specific Real-Time PCR machine to obtain the limit of detection limit and the upper and lower detection limit.
However, you can use only one extraction kit, and one PCR kit and one machine and optimize your reaction.
The extraction is the most important step so, you can try the different extarction kits you have (but by using the same PCR kit and machine), to select the one giving highest yield of RNA and dont forget the positive and negative contril in run.
If the kit dosen’t has standards that should be run and measured with each run, you generate your own, by doing convential PCR for known concentration of extracted RNA at 260nm, and then run on normal agarose gel, purify your band from the gel, measure its OD and then do your standard curve by serial dilution and then do a run for these dilutions on the Real time to generate the standard curve, and then use it in your calculations.
To my knowelege, the RNA absolute quantification is done by one of these two methods, plasmide provides by the kits contains the region of interest or do it through amplification and gel …..etc
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