Detection of mycoplasma contaminations by the polymerase chain reaction

• Published in: Cytotechnology (Dordrecht) Vol. 16; no. 2; pp. 67 - 77
• Main Authors: WIRTH, M, BERTHOLD, E, GRASHOFF, M, PFÜTZNER, H, SCHUBERT, U, HAUSER, H
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer 01-01-1994
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Base Sequence; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Genetic Code; Genetic engineering; Genetic technics; Methods. Procedures. Technologies; Microbiology; Miscellaneous; Molecular Sequence Data; Mycoplasma - isolation & purification; Mycoplasma arginini; Mycoplasma fermentans; Mycoplasma hyorhinis; Polymerase Chain Reaction; RNA, Ribosomal, 16S - genetics; Sensitivity and Specificity; Synthetic digonucleotides and genes. Sequencing; Time Factors; Nucleotide sequence; Acholeplasma; Sequence alignment; Primer; Homology; Acholeplasmataceae; Method; Mycoplasma; Mycoplasmataceae; Mollicutes; Polymerase chain reaction; Mycoplasmatales; Ribosomal RNA; Pathogenic; Acholeplasmatales; Microorganism culture; 16S-RNA; Bacteria; Detection; Comparative study
• ISSN: 0920-9069; 1573-0778
• DOI: 10.1007/bf00754609

The polymerase chain reaction (PCR) has been used for the general detection of Mollicutes. 25 Mycoplasma and Acholeplasma species were detected including important contaminants of cell cultures such as M. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1-2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.