Oligonucleotide probes complementary to 16S rRNA for rapid detection of mycoplasma contamination in cell cultures

FEMS Microbiology Letters 107 (1993) 139-144 © 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00 Published by Elsevier 139 FEMSLE 05291 Jens G. Mattsson and Karl-Erik Johansson The National Veterinary Institute, Uppsala, Sweden (Received 9 November 1992; accepted 30 November 1992) Abstract: Mycoplasma contamination of cell cultures is a menace to diagnostic and research procedures. Rapid and reliable detection methods are, therefore, sorely needed. After comparing 16S rRNA sequences from those mycoplasmas that contaminate cell cultures, three different oligonucleotide probes were constructed. Two of these probes were designed to be group-specific and one to be species-specific. The three oligonucleotide probes were designed to cover all mycoplasmas commonly isolated from cell cultures. Contaminated cell lines could easily be detected by a direct filter hybridization assay in which the probes were incubated jointly. The assay proved to be rapid and sensitive with the possibility to perform and evaluate the mycoplasma testing within one working day. Key words: Cell culture; Hybridization; Mycoplasma; Mycoplasma contamination; Probe; rRNA Introduction Today a wide range of biomedical research laboratories as well as hospitals and biotechnical industries are becoming more and more dependent on procedures where ceils are grown in vitro. The applications vary from studies of cell proliferation and gene regulation to testing and production of biologically active substances. Fu- Correspondence to: Kari-Erik Johansson, Laboratory of Bacteriology, The National Veterinary Institute, Box 7073, S-750 07 Uppsala, Sweden. ture increases in the use of cell cultures can also be expected as the utilization of laboratory animals becomes more restricted due to animal protection laws. The importance of good cell culture conditions can not be over-emphasized, if accurate and reproducible results are to be obtained. Bacterial and fungal contaminations may cause some concern, but are usually obvious and easily detected. A much more serious problem is the insidious infections caused by mycoplasmas [1,2]. The presence of these wall-less prokaryotes, which are difficult to detect and even more difficult to eradicate, often leads to erroneous results or causes the loss of unique cell lines. Many reports describe deleterious consequences of mycoplasma Oligonucleotide probes complementary to 16S rRNA for rapid detection of mycoplasma contamination in cell cultures 140 Materials and Methods screened using the oligonucleotide probes. The species of the contaminating mycoplasma was determined by immunofluorescence [16]. All but one of the cell lines analyzed were authentic samples originally submitted to the Mycoplasma laboratory at our institute for mycoplasma assays. This cell line (Vero), which had been experimentally infected with Mycoplasma pirum (ATCC 25969), was used as a sample in the assay shown in Fig 2. Synthesis and labelling of oligonucleotides Sequences appropriate for the construction of probes were identified using the program package from the Genetics Computer Group at the University of Wisconsin [18]. The oligonucleotides were synthesized on a DNA synthesizer, model 391, from Applied Biosystems (Foster City, CA, USA) and labelled at the 5' end with [3'32p]ATP using T4 polynucleotide kinase. The different oligonucleotides were either labelled separately or as a cocktail. DNA-RNA filter hybridization Cell culture samples were applied onto nylon membranes (Zeta-probe, Bio-Rad, Richmond, CA, USA) and the rRNA was made accessible for hybridization as described earlier [12]. The prehybridization was carried out at 59°C for 30 min in a solution of 2 x SSC, 10 x Denhardt's solution, 2% sodium dodecyl sulphate (SDS) and denatured herring sperm DNA (0.15 mg/ml). The oligonucleotide probes were added to the prehybridization solution (1 x 106 epm/ml) and the incubation was continued for 90 rain. After completion of the hybridization the filters were rinsed once for 5 rain at 59°C in 2 x SSC and 3 X 10 min in 2 × SSC at 49°C. Hybridization signals were detected by autoradiography for 2-6 h on Kodak X-Omat AR films (Eastman Kodak Co, Rochester, NY, USA). Cell cultures Mycoplasma-free indicator cell lines (mouse embryo fibroblast 3T6 cells and Vero monkey kidney cells) were used as negative controls. Cell lines assayed for mycoplasma by microbiological culture [16], fluorescent DNA staining [17] and hybridization with the H900 probe [12] were Results and Discussion Sequence alignment The most frequently occurring mycoplasmas contaminating cell cultures are Mycoplasma infections in cell cultures. For instance, false negative results during HIV isolations [3], increased invasive behaviour of tumour cells [4] and new immunosuppresive properties thought to be produced by thymus-derived T-cells [5] have been reported. Numerous methods for detecting mycoplasma infection have been developed [6]. Normal microbiological culture of mycoplasmas is mainly restricted to special laboratories, due to the fastidious nature of mycoplasmas. Furthermore, some strains of Mycoplasma hyorhinis are possible to cultivate only under highly specialized conditions [7,8]. Indirect methods are, therefore, recommended not only as alternatives but also as a complement to cultivation. Methods are described for the biochemical detection of mycoplasmas, based on differences in eubacterial and eukaryotic metabolism or the use of fluorescent DNA staining [1,6]. Immunological methods have also been used for analysis of cell cultures [9]. All of these methods are time-consuming and their use is fraught with difficulties. An approach that has been recognized as a powerful alternative to classical methods is to utilize assays based on nucleic acids hybridization [10-12]. The probes used, as well as a commercial kit from GenProbe ® (San Diego, CA) [13,14], are based on restriction fragments or cDNA complementary to rRNA. The use of oligont,cleotide probes for group specific detection of mycoplasmas has been reported, but the specificities of these probes were not satisfactory due to lack of adequate sequence information [15]. The present report describes the detection of mycoplasma contamination using a set of short oligonucleotide probes complementary to 16S rRNA. 141 arginini, Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma hyorhinis, Mycoplasma orale and Acholeplasrna laidlawii [1,16]. Of these six species, A. laidlawii belongs to the anaeroplasma group and the rest to the hominis group according to Weisburg et al. [19], who have arranged the mycoplasmas into five distinct phylogenetic groups. Through alignment of published 16S rRNA sequences [19,20] we were able to identify a region that is unique for all mycoplasmas from the hominis group and hence a complementary oligonucleotide, designated Mccl, was constructed (Fig. la). However, it was not possible to design an oligonucleoti (...truncated)