While various methods exist for the detection of Mycoplasma contamination [13, 14], probably the most frequently used ones are biochemical detection of Mycoplasma metabolism and PCR-based detection of Mycoplasma DNA. Though the biochemical detection of mycoplasma ATP generation (Mycoalert (Lonza, Basel, Switzerland)) is a quick protocol, it has certain disadvantages that should be mentioned, including requiring that reagents be reconstituted and brought to 22oC before each measurement and requiring a luminometer for ATP detection. Aspecificity due to ATP generated by other cells may lead to a high background and eventually false negative measurements. The Ureaplasma species which are also a common contaminant in a cell culture  cannot be detected by Mycoalert as their own ATP production relies on the hydrolysis of urea . Finally, the sensitivity of biochemical detection has been shown to be lower than that for PCR or qPCR methods [17, 18].
There are a variety of kits on offer based on regular PCR, followed by gel electrophoresis. The major advantage of these kits is the wide availability of regular PCR and electrophoresis equipment. However, decreased specificity compared to probe-based qPCR, the additional electrophoresis step, and the inability to quantitatively monitor the decrease in Mycoplasma genome concentration during treatment are clear drawbacks. Intercalation-based (e.g. SYBR Green) qPCR kits such as MycoSEQ Mycoplasma Detection Assay (Thermo Fisher, Waltham, MA, USA) eliminate the electrophoresis step and provide quantitative information about Mycoplasma genome concentration. The disadvantages of intercalation-based qPCR kits compared to probe-based kits are a lower specificity, lack of internal control and the potential effect of cell culture composition, ionic composition and ionic strength to change the melting temperature of the qPCR product [19–21]. Since this melting temperature is the basis for evaluating specificity in intercalation based qPCRs, changing it can be problematic. Probe-based qPCRs such as PhoenixDx (Procomcure Biotech, Thalgau, Austria), Microsart RESEARCH Mycoplasma (Sartorius, Goettingen, Germany) and qPCR Detection Kit (XpressBio, Frederick, MD, USA) avoid these problems and due the additional requirement of the binding of the probe sequence, these kits provide a higher specificity than regular PCRs and intercalation-based qPCRs.
Noting the advantages of probe-based qPCRs, we optimized the Procomcure PhoenixDx kit to perform a direct qPCR with a Mycoplasma infected U937 cell culture. Our results indicates that the optimal temperature was the same as that in the original protocol, so the primer + probe binding was not affected by the presence of the direct template. The fact that the optimal template volume was 6 ml (30% of the total qPCR volume) meant that the direct sample did not have a significant inhibitory effect on the qPCR. A major optimization step that we performed was decreasing the annealing/extension time from 60 sec to 20 sec, thus saving 40 seconds in each cycle. Interestingly, this decrease led to only a minor decrease in the sensitivity (~0.6 Ct level increase). In addition, decreasing the number of cycles from 50 to 40, reduced the total qPCR time required to 65 minutes. When we used the optimized qPCR protocol with direct and purified cell culture templates, we found that Ct levels of a 6 ml direct template was almost identical to that of purified DNA from a 60 ml cell culture. The reason for this is mainly due to a dilution of the original DNA content during the elution step at the end of DNA purification. Overall in our case, direct qPCR sensitivity was higher than qPCR with a purified template, with a saving in the cost/time of DNA purification. We monitored the elimination of Mycoplasma contamination from the U937 cell culture using the optimized direct qPCR protocol. One of the concerns using pathogen DNA detection is that the non-viable pathogen’s DNA can also be detected and lead to a false positive signal. In our case however, the Mycoplasma DNA content dropped to ~20% of the original concentration after 1 day of treatment, and though days 1 and 2 contained a similar level of DNA, this decrease continued on day 3. In summary, with direct qPCR we were able to monitor the elimination of Mycoplasma over the treatment period.
In conclusion, we optimized a probe-based qPCR to detect Mycoplasma contamination in a user-friendly manner. This direct qPCR method does not require a purification step, maintains sensitivity and offers a shorter 65 minute protocol.
While we did not observe a major qPCR inhibitory effect of U937 cell culture, it cannot be ruled out that components of other cell cultures may have an inhibitory effect. Most probe based qPCR kits, including the kit used here, contain an internal control (e.g. HEX-labelled probe), therefore the detection of qPCR inhibition (no FAM, no HEX signals) is straightforward. In the case of qPCR inhibition, dilution of the direct sample may be a solution for decreasing/eliminating qPCR inhibition.