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

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.

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

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.

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

Objective We aim to directly detect Mycoplasma DNA in a U937 suspension cell culture without using DNA purification. In order to make Mycoplasma contamination monitoring easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit. We compared the sensitivity of direct qPCR against qPCR with a purified DNA template. Results Our findings indicate that qPCR worked optimally with a 6 μl sample volume and a 52 °C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 to 20 s without any major decrease in reaction sensitivity. The total cycle time of optimized direct qPCR was 65 min. The optimized qPCR protocol was used to detect Mycoplasma DNA before and after DNA purification. Our findings indicate that direct qPCR had a higher sensitivity than regular qPCR. Ct levels produced by direct qPCR with 6 μl templates were almost identical to Ct levels produced by regular qPCR with DNA purified from a 60 μl 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 U937 cell cultures.

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

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.