by Stephanie Schweizer and Alexandra Scholz
Life Science
A Matter of Speed: Real-Time PCR for the Rapid Detection of Mycoplasma Contamination
ycoplasma are among the world’s smallest bacteria capable of inde- pendent reproduction. They belong to the class of Mollicutes and have a very slow and parasitic growth. They cause numerous infections in animals and plants.
M
Mycoplasma are very difficult to control be- cause they lack the bacterial cell wall, which is the main point of attack for many antibiot- ics. 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., cultivation in special liquid or solid nutrient media, or detection by means of fluorescence microscopy—are very com- mon and in combination represent the “gold standard.” Growth-based detection requires a cultivation time of at least 28 days before con- tamination 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 fluores- cence 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.
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
Figure 2 – Operation of Microsart AMP Mycoplasma Kit.
AMERICAN LABORATORY • 9 • JUNE/JULY 2014
Figure 1 – Microsart AMP Mycoplasma Kit from Sartorius.
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 prepa-
ration with a Vivaspin 20 unit with subsequent DNA isolation and Microsart AMP mycoplasma detection. Mycoplasma fermentans were culti- vated 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.
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 concen- trated 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 concen- trated sample and 200 μL lysis buffer, was incubated for 15 min at 56 °C to complete the cell lysis. Following this, the samples under- went 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 pre- pared as specified in the kit manual according to the following protocol:
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