Realtime 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 many infections in animals and plants.

Mycoplasma are very difficult to control as 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. 

Detection of Mycoplasma

There are many methods for the identification of a mycoplasma contamination. Growth-based methods, e.g. the cultivation in special liquid or solid nutrient media, or the detection by means of fluorescence microscopy are very common and in combination represent the „golden standard“. Growth-based detection requires a cultivation time of at least 28 days before a 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 great 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 lot of experience and specific know-how for the interpretation of the results. 

The 3-Hour Alternative

PCR-based detection kits such as the Microsart AMP Mycoplasma Kit from Sartorius offer their users a sensitive and robust detection within only three hours. The method is simple and costeffective; the kit is supplied ready for use. All that is needed on top is a Realtime 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, a volume of up to 18 ml can be processed, which ensures an increase in sensitivity. 

Material and Methods

The following describes the example of sample preparation with a Vivaspin 20 unit with subsequent DNA isolation and Microsart AMP mycoplasma detection. Mycoplasma fermentans were cultivated in a Hayflick medium at 37 ÅãC until a slight color change was apparent of the phenol red indicator and then diluted 1:1,000 in 1 x PBS (Phosphate Buffered Saline). 18 ml of the diluted sample were pipetted into each of the Vivaspin 20 units. In order to neutralize non-specific compounds, 2 ml of Coating Buffer were added to each of the samples. This was followed by a centrifugation phase of 3,500 x g for 20 minutes, until the concentrated retentate volume was approximately 200 μl. With the aid of 200 μl lysis buffer, the entire concentrate was transferred into a new 1.5 ml reaction tube. The buffer was used to flush the Vivaspin units in order to guarantee a 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 ÅãC in order to complete the cell lysis. After 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 Realtime 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 in order to enable a direct comparison between the samples, with and without concentration. The PCR reactions were prepared as specified in the kit manual, according to the following protocol:

• Centrifuging of the reaction tubes contained in the kit with the lyophilized components for 5 seconds at maximum speed. Addition of 1,275 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 incubation at room temperature until rehydration is complete.
• Brief mixing of the rehydrated solutions with the Vortex mixer and 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 pre-programmed Realtime Thermoycler.

The results are illustrated in Table 1 and 2 and in Figure 1. The Ct value (Cycle Threshold) of the concentrated samples was always compared with samples which 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-Ct value resulting from Table 1 and the Ct difference which results from using or not using the Coating Buffer. The delta Ct value is determined as follows:

Δ Ct = Ct (sample without Vivaspin concentration) – Ct (concentrated sample)

With the concentration in combination with the Coating Buffer a delta-Ct value of Δ Ct > 7 could be achieved for Mycoplasma fermentans. When the Coating Buffer was not used (individual values are not listed) a lower delta-Ct value of Δ Ct ~ 6 was recorded although it was possible to profit from the shorter centrifuging times of the Vivaspin units. However, this still represents a considerable increase in sensitivity.

Parameters such as sensitivity and time saved are essential for the investigation of mycoplasma contamination. These are fulfilled with the described PCR Kit in combination with the concentration step. By using Vivaspin 20, a Ct improvement of Δ Ct > 7 could be achieved, which corresponds to an increase in sensitivity of approximately 2 log levels. This makes the Microsart AMP Mycoplasma Detection Kit and Vivaspin 20 a cost-effective and practical quick-test for mycoplasma.

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