Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

Abstract

Objective In order to make the Mycoplasma contamination monitoring task easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit to detect Mycoplasma DNA without DNA purification in a U937 suspension cell culture. We compared the sensitivities of the direct qPCR and the qPCR with the purified DNA template. Results Our findings indicate that qPCR worked optimally with a 6 ml sample volume and a 52 o C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 sec to 20 sec without any major decrease in the reaction sensitivity. The total cycle time of the optimized direct qPCR was 65 minutes. The optimized qPCR protocol was used to detect Mycoplasma DNA directly and after DNA purification. Our findings indicate that the direct qPCR had a higher sensitivity compared to the regular qPCR. The Ct levels produced by the direct qPCR with 6 ml templates were almost identical to the Ct levels produced by the regular qPCR with DNA purified from a 60 ml cell culture sample (23.42 vs 23.49 average Ct levels respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from the U937 cell cultures.