A Matter of Speed: Real-Time PCR for the Rapid Detection of Mycoplasma Contamination

Mycoplasma are among the world’s smallest bacteria capable of independent reproduction. They belong to the class of Mollicutes and have a very slow and parasitic growth. They cause numerous infections in animals and plants.

Mycoplasma are very difficult to control because they lack the bacterial cell wall, which is the main point of attack for many antibiotics. For this reason, a complete retention with conventional cell structure sterile filters (0.2 μm pore size) is not possible. The danger is that the mycoplasma with a cell size between 0.5 and 0.8 μm could pass through the pores due to their great flexibility and capacity for deformation.

Figure 1 – Microsart AMP Mycoplasma Kit from Sartorius.

Detection of mycoplasma

There are many methods for the identification of a mycoplasma contamination. Growth-based methods—e.g., cultivation in special liquid or solid nutrient media, or detection by means of fluorescence microscopy—are very common and in combination represent the “gold standard.” Growth-based detection requires a cultivation time of at least 28 days before contamination with these slow-growing bacteria can be ruled out with certainty. During this period, the sample must be visually inspected on a daily basis, which requires a significant amount of time. Even with the aid of fluorescence detection, it takes at least eight days before a mycoplasma infection can be ruled out. In addition, the user needs a trained eye, a great deal of experience, and specific knowlege in order to interpret the results.

Figure 2 – Operation of Microsart AMP Mycoplasma Kit.

Three-hour alternative to mycoplasma identification

PCR-based detection kits such as the Microsart AMP Mycoplasma Kit from Sartorius (Göttingen, Germany) (Figure 1) offer users sensitive and robust detection in only 3 hr. The method is simple and cost effective; the kit is supplied ready for use (Figure 2). All that is needed on top is a real-time thermocycler that is capable of detecting the fluorescent dyes FAM and ROX. The PCR Kit is suitable for a wide variety of initial matrices. In combination with a Vivaspin 20 or Vivaspin 6 ultrafiltration unit (Sartorius), a volume of up to 18 mL can be processed, which ensures increased sensitivity.

Materials and methods

The following is an example of sample preparation with a Vivaspin 20 unit with subsequent DNA isolation and Microsart AMP mycoplasma detection. Mycoplasma fermentans were cultivated in Hayflick medium at 37 °C until a slight color change was apparent in the phenol red indicator; the sample was then diluted 1:1000 in 1× PBS (phosphate buffered saline). Eighteen milliliters of the diluted sample was pipetted into each of the Vivaspin 20 units.

Table 1 – Ct values of the samples with and without concentration step (RFU = relative fluorescence units)

In order to neutralize nonspecific compounds, 2 mL of coating buffer was added to each of the samples. This was followed by a centrifugation phase of 3500 × g for 20 min, until the concentrated retentate volume was approximately 200 μL. With the aid of 200 μL lysis buffer, the entire concentrate was transferred to a new 1.5-mL reaction tube. The buffer was used to flush the Vivaspin units to ensure complete quantitative transfer of the sample.

The mixture, consisting of 200 μL concentrated sample and 200 μL lysis buffer, was incubated for 15 min at 56 °C to complete the cell lysis. Following this, the samples underwent DNA isolation with the use of silica-based centrifuge tubes.

During the DNA isolation, the Microsart AMP Extraction Kit was used according to the kit manual. Almost the entire isolated DNA was used in the real-time PCR (50 μL of the 60 μL DNA eluate) in order to achieve maximum sensitivity. In parallel, the DNA of the same initial material was isolated without the Vivaspin concentration step to enable a direct comparison of the samples with and without concentration. The PCR reactions were prepared as specified in the kit manual according to the following protocol:

Table 2 – ΔCt values with and without the use of coating buffer
    • Centrifugation of the reaction tubes contained in the kit with the lyophilized components for 5 sec at max. speed. Addition of 1275 μL rehydration buffer to the primer/probe/nucleotide mix (red cap).
    • Addition of 300 μL water (PCR quality) to the positive control (green cap) and to the internal control (yellow cap).
    • Five minutes of incubation at room temperature until rehydration was complete.
    • Brief mixing of the rehydrated solutions with the vortex mixer and short centrifugation.
    • Composition of the master mix: per reaction, pipetting and mixing of 49 μL primer/probe/nucleotide mix (red cap) and 1 μL of the internal control (yellow cap) into a 1.5-mL reaction tube. 
    • Pipetting of 50 μL of the master mix plus 50 μL of the sample or positive or negative control material (e.g., elution buffer, PCR water, or 10 mM tris buffer) into 200-μL PCR tubes and insertion into the preprogrammed real-time thermoycler.
Figure 3 – Amplification curves for Mycoplasma fermentans samples with and without previous Vivaspin concentration step, including coating buffer.

The results are illustrated in Tables 1 and 2 and Figure 3. The Ct (cycle threshold) value of the concentrated samples was always compared with samples that were not concentrated. The Ct value is the amplification cycle at which a certain fluorescence threshold value is exceeded. Table 1 lists the individual Ct values. Table 2 shows the average delta (Δ) Ct value from Table 1 and the Ct difference, which results from using or not using the coating buffer. The ΔCt value is determined as follows: ΔCt = Ct (sample without Vivaspin concentration) – Ct (concentrated sample).

With the concentration in combination with the coating buffer, a ΔCt value of >7 could be achieved for Mycoplasma fermentans. When the coating buffer was not used (individual values are not listed) a lower ΔCt value of ~6 was recorded, although it was possible to take benefit from the shorter centrifugation times of the Vivaspin units. However, this still is a considerable increase in sensitivity.

Conclusion

Parameters such as sensitivity and time savings are essential for the investigation of mycoplasma contamination. This is accomplished with the PCR Kit in combination with the concentration step described above. By using Vivaspin 20, a ΔCt improvement >7 can be achieved, which corresponds to an increase in sensitivity of approx. 2 log levels. This makes the Microsart AMP Mycoplasma Detection Kit and Vivaspin 20 a cost-effective, practical, and rapid test for mycoplasma.

The authors are with Sartorius AG, August-Spindler-Strasse 11, 37079 Göttingen, Germany; tel.: +49 551 3080; fax: +49 551 308 3289; e-mail: info@ sartorius.com www.sartorius.com

Comments