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

摘要

Optimization of genus-specific direct qPCR and comparison of its performance with regular qPCR using purified DNA templates. Effect of the qPCR annealing/extension temperature on the direct qPCR performance. Student’s test was applied to compare the Ct values of samples with various annealing/extension temperatures to those samples with a 50 °C annealing/extension temperature (n = 3). Effect of the annealing/extension time on direct qPCR performance. Student’s t-test was applied to compare the Ct values of the samples with various annealing/extension times to those samples with a 60 s annealing/extension time (n = 4). Effect of sample volume on direct qPCR performance. Student’s t-test was applied to compare the Ct values of various template volume samples with samples having a 10 μl template volume (n = 3). Comparison of direct qPCR performance with regular qPCR using a purified DNA template (n = 3). The DNA was purified from a 6, 60 and 120 μl cell culture supernatant via the QIAamp protocol and eluted in a 100 μl elution buffer. 6 μl of eluted DNA was used in the qPCR procedure. As a comparison, 6 μl of the cell culture supernatant was used in direct qPCR. NA: no amplification was detected. * P