We have made a novel technical breakthrough in qPCR application by improving the sensitivity to 8 copies/ug DNA in 20μL reaction. The method is robust and consistent and has been validated internally and will be used for future gene and cell therapy contract studies. For instance, we applied this method to VSV-G qPCR for RCL detection in Car-T and related studies, which can monitor RCL activity in 1μg of genomic DNA or 145000 human cells in 20μL reaction. This technical improvement significantly increases the scope of qPCR application in pharmacokinetics study of drugs of gene and cell therapy. In the presentation, the author will elaborate on the detailed processes of how this innovative method has been developed and validated. The current FDA guidance on Quantitative Polymerase Chain Reaction (qPCR) method requires reported lower limit of quantitation (LLOQ) to be less than 50 copies of a target of interest per microgram of total DNA input. It is widely recognized in both academia and industry that excessively high concentrations (> 500-1000 ng/100 μL) of starting DNA template can inhibit amplification reactions, due to carryover inhibitors or inefficient denaturation. Due to this reason, 10 to 100 ng of DNA template per 100 μL reaction volume is commonly recommended. However, because the LLOQ is reported against 1000 ng of DNA input, the reported LLOQ from reactions that utilize only 100 ng of DNA template must be corrected upwards 10-fold. Consequently, to follow the current FDA guidance, a 100 µL qPCR reaction using 100 ng of DNA template must satisfy the general 20% CV and 20% RE criteria from BMV guidelines when the nominal concentration of the target is only 5 copies / reaction, a difficult task using current technologies. This task becomes nearly impossible if the more common 20 µL qPCR reaction is utilized instead, as it would require the reported LLOQ to be 1 copy / reaction.
To address this popular problem, we developed and validated a qPCR method by employing a high concentration of enzyme digested genomic DNA (1000ng) in 20 μL qPCR reaction, which is generally considered to be an unfavorable condition for amplicon amplification. Surprisingly, we found that such high levels of human genomic DNA background in qPCR reaction can make the reaction more robust against changes in amplification efficiency and stabilize data reproducibility for the amplification of both the target gene and a control gene in a multiplex qPCR reaction. Moreover, our protocols maintain an accuracy and precision very well within BMV guidelines even when using extremely low concentrations of the target vector, with a reported LLOQ as low as 8 copies of target gene per 1 μg of DNA template (and with no correction factor necessary). The reported %CV and % RE are generally less than 2%. Thus, we assert that this optimized qPCR method represents a significant technical breakthrough and could mark the start of a new chapter for qPCR amplification applications. The long-term technical problem of qPCR LLOQ sensitivity has been solved by our approach.
Learning Objectives:
Upon completion, participant will be able to learn a novel qPCR method that quantitate VSV-G gene copy (RCL activity) and use it in their own research and development.
Upon completion, participant will be able to have a chance to discuss with the presenter on all the experimental details to establish similar qPCR assay.
Upon completion, participant will be able to have a chance to discuss with the presenter on other qPCR and qPCR applications like RCL, Stem-loop qPCR, multiplex qPCR/ddPCR, etc.