Materials and methods
Cell culture
Mycoplasma infected U937 human monocytic cells were grown in an RPMI 1640 medium containing 10% heat-inactivated FBS (Sigma, St. Louis, MO, USA), and 50 μg/mL gentamicin at 37 °C in 5% CO2, all within a 25 cm2 cell culture flask (Greiner Bio-One Hungary, Mosonmagyaróvár, Hungary).
Mycoplasma elimination
Mycoplasma elimination was performed using Mycoplasma Elimination Reagent (Bio-Rad, Hercules, CA, USA). The reagent was added to the RPMI 1640 medium at a 0.5 μg/ml final concentration and the U937 cells were then cultured in this medium for 7 days.
DNA extraction and qPCR
DNA was extracted from Mycoplasma infected U937 cell supernatants using the Qiagen QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. PhoenixDx® Mycoplasma Mix (Procomcure Biotech, Thalgau, Austria) was used in the qPCR experiments. qPCRs with 20 μl final volume were performed using the Bio-Rad CFX Connect qPCR real-time system. A statistical comparison of qPCR cycle threshold (Ct) values was performed with Student’s t test, as described previously [12].
Results
To achieve optimal sensitivity and the shortest possible reaction time of direct qPCR, we followed a step-wise optimization of the PhoenixDx Mycoplasma Mix (Procomcure Biotech, Thalgau, Austria) protocol that was originally designed to amplify purified DNA samples. First, we tested the optimal annealing/extension temperature for detecting unpurified Mycoplasma DNA in Mycoplasma-infected U937 cell culture supernatants (Fig. 1a). The results indicated that reactions with 50–52 °C annealing/extension temperature produced the lowest Ct values (26.84 ± 0.14–27.06 ± 0.26). We chose the 52 °C annealing/extension temperature for further tests. Next, we tested to see whether reducing the annealing/extension time might influence qPCR performance (Fig. 1b). Our findings showed that the 60 s annealing/extension time provided the lowest Ct values (23.56 ± 0.47), but the 20 and 40 s annealing/extension times led to only slightly higher Ct values (24.20 ± 0.23, 24.11 ± 0.27, respectively), which suggested that reducing the annealing/extension time from 60 to 20 s had a minimal influence on qPCR sensitivity. 20 s annealing/extension time was used for further qPCRs. Next, we tested the effect of sample volume on qPCR performance (Fig. 1c). The Ct levels of samples with 6 μl, 8 μl and 10 μl volumes of supernatants were similar (21.92–22.13 Ct value range), indicating that qPCR sensitivity is influenced by higher Mycoplasma DNA content and also by a higher level of qPCR inhibition in the 8 and 10 μl samples. In further experiments, we opted for the 6 μl sample volume. Finally, we compared the performance of direct qPCR and regular qPCR with purified DNA samples (Fig. 1d). The QIAamp DNA purification kit was used to isolate Mycoplasma DNA from U937 cell cultures (medium + cells). The elution volume was 100 μl. A comparison of the 6 μl direct sample volume and 6 μl purified sample was not possible as just 6 μl of the 100 μl total elution volume could be used during regular qPCR. Therefore we also decreased the 6 μl direct sample volume by a factor of 6/100 (0.36 μl). In a comparison of these samples we found that the 6 μl purified sample produced lower Ct values (~ 2 cycles) than the 0.36 μl direct sample, suggesting a low level of qPCR inhibition of the supernatant. However, when we compared the Ct levels of samples with 6 μl supernatant to the Ct levels of samples with purified DNAs we noticed that the Ct values produced with 6 μl supernatants were almost identical to those of the purified 60 μl supernatant (23.42 ± 0.26, 23.49 ± 0.30, respectively) indicating an altogether higher sensitivity of the direct qPCR.
As an application of optimized direct qPCR we monitored Mycoplasma elimination from the infected U937 cell culture. Our results showed that the supernatants (n = 4) containing removal agent or free from removal agent both resulted in nearly the same Ct levels (27.04 ± 0.24 and 26.94 ± 0.45, respectively) (Fig. 2a). This indicated that the presence of removal agent did not influence qPCR performance. Mycoplasma DNA dropped rapidly (by ~ 80%) after a 24-hour treatment (Fig. 2b). On the fourth day, Mycoplasma concentration was 2.3% of the original concentration. By the sixth day of treatment, Mycoplasma DNA was no longer detectable (data not shown). Overall, direct qPCR method proved to be a quick and effective method for monitoring the decrease in Mycoplasma DNA during the elimination process.
Discussion
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 22 °C 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 [15] cannot be detected by Mycoalert as their own ATP production relies on the hydrolysis of urea [16]. 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 [19,20,21]. 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 μl (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 s to 20 s, thus saving 40 s 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 min. When we used the optimized qPCR protocol with direct and purified cell culture templates, we found that Ct levels of a 6 μl direct template was almost identical to that of purified DNA from a 60 μl 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 min protocol.