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FEMS Microbiology Letters May 1995Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed...
Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumoniae. To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.
Topics: Base Sequence; DNA Primers; DNA, Bacterial; Molecular Sequence Data; Mycoplasma; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Reference Standards
PubMed: 7750739
DOI: 10.1111/j.1574-6968.1995.tb07524.x -
Journal of Cellular Physiology Jan 1983The aliphatic diamine putrescine, a metabolic precursor of the polyamines spermidine and spermine, markedly stimulated the growth of a murine lymphoblastoid cell line (R...
The aliphatic diamine putrescine, a metabolic precursor of the polyamines spermidine and spermine, markedly stimulated the growth of a murine lymphoblastoid cell line (R 1.1) infected with Mycoplasma orale, under conditions of arginine limitation. The diamine acted by suppressing the growth of the mycoplasma, which use arginine as a major energy source, and thereby prevented the depletion of arginine from the medium. The antimycoplasmal effects of putrescine occurred at concentrations that were neither stimulatory nor toxic to uninfected cells.
Topics: Animals; Arginine; Cell Division; Cell Line; Mice; Mycoplasma; Mycoplasma Infections; Putrescine
PubMed: 6826657
DOI: 10.1002/jcp.1041140104 -
Nucleic Acids Research Mar 1980The DNAs of four Mycoplasma and one Acholeplasma species were found to contain methylated bases. All of the five species contained 6-methyladenine (m6Ade), the... (Comparative Study)
Comparative Study
The DNAs of four Mycoplasma and one Acholeplasma species were found to contain methylated bases. All of the five species contained 6-methyladenine (m6Ade), the methylated base characteristic of prokaryotic DNA. The extent of methylation of adenine residues in the mycoplasmal DNA ranged from 0.2% in Mycoplasma capricolum to about 2% in Mycoplasma arginini and Mycoplasma hyorhinis with intermediate methylation values for Mycoplasma orale and Acholeplasma laidlawii DNAs. About 5.8% of the cytosine residues in M. hyorhinis DNA were methylated also. Analysis of cell culture DNA for the presence of m6Ade as a means for detection of contamination by mycoplasmas, and the phylogenetic implications of the finding of methylated bases in mycoplasmal DNAs are discussed.
Topics: 5-Methylcytosine; Acholeplasma laidlawii; Adenine; Base Composition; Cytosine; DNA, Bacterial; Methylation; Mycoplasma
PubMed: 7433124
DOI: 10.1093/nar/8.6.1383 -
Radiation Research Sep 1991Mycoplasma infection of mammalian cells in culture is a common occurrence that can affect the results of experimental protocols. Current methods of eliminating...
Mycoplasma infection of mammalian cells in culture is a common occurrence that can affect the results of experimental protocols. Current methods of eliminating mycoplasma from cell cultures are usually tedious, time-consuming, and sometimes unsuccessful. In the present study, four cultured brain tumor cell lines (human U-251 MG, U-87 MG, SF-126, and rat 9L) were heavily contaminated with Mycoplasma orale. Heating the cultures to 41 degrees C for at least 96 h eliminated the contamination for up to 7 months, the maximum period of observation. The time chosen to assay for the presence of mycoplasma in cultures was critical: in some cultures heated for less than 96 h that initially appeared to be free of contamination, mycoplasma began to appear after 2 weeks. Heat-treated cells grew at the same rate as unheated control cells. Infected cells were more sensitive to X rays than uncontaminated cells, but the sensitivity reverted to normal after mycoplasma was eliminated by hyperthermia. The heating method does not require a cell cloning procedure or the use of exogenous materials. Treated cell cultures exhibit normal growth and radiation sensitivity, and the technique seems to be reliable and efficient.
Topics: Animals; Brain Neoplasms; Hot Temperature; Humans; Mycoplasma; Rats; Time Factors; Tumor Cells, Cultured
PubMed: 1886990
DOI: No ID Found -
Journal of Immunological Methods Nov 1988Mycoplasmas are a major cause of cell culture contamination and are especially troublesome during HAT selection. The enzyme adenosine phosphorylase (adoP) is present in...
Mycoplasmas are a major cause of cell culture contamination and are especially troublesome during HAT selection. The enzyme adenosine phosphorylase (adoP) is present in all common mycoplasma species but is considered to have a low activity in mammalian cells. However, using an adoP screening test, we have observed that some cell cultures do possess an intrinsic adoP activity leading to false positive results. Moreover, as a false negative result, we encountered a variant of Mycoplasma orale (identified after cultivation on agar and immunostaining) which was not detectable with the adoP screening in cell culture supernatants and only at low levels in cell lysates. To increase the low signal/noise adoP ratio found there, we used an indicator cell line with low intrinsic activity. Indicator cells were inoculated with the test supernatant and the adoP activity of these infected cells were measured after lysis. The procedure diminished the effect of biological variation in intrinsic enzyme activity between the several cell lines tested. Furthermore, in another mycoplasma infected cell line (with M. fermentans), this infection was only reliably detected using these indicator cells. With this procedure we obtained rapid results which were concordant with those obtained using the time consuming cultivation on agar.
Topics: Animals; Biomarkers; Cell Line; Cell-Free System; Fibroblasts; Mice; Mycoplasma; Pentosyltransferases; Purine-Nucleoside Phosphorylase
PubMed: 3141514
DOI: 10.1016/0022-1759(88)90162-7 -
Journal of Medical Microbiology Mar 1990All of five lyophilised cultures of Mycoplasma orale kept for 23 years at room temperature were still viable, as were all but one of 12 lyophilised cultures of six...
All of five lyophilised cultures of Mycoplasma orale kept for 23 years at room temperature were still viable, as were all but one of 12 lyophilised cultures of six Mycoplasma spp. that had been stored for 18-22 years at 4 degrees C. Similarly, 11 of 13 lyophilised ureaplasma cultures were viable after 8-22 years at 4 degrees C; the titre of organisms in the viable cultures had diminished no more than 100-fold. Seven broth cultures of five different Mycoplasma spp. all proved viable 5-13 years after being frozen and stored at -70 degrees C, although there was up to 10(4)-fold reduction in the titre of organisms in some cultures. Furthermore, 18 (82%) of 22 different Mycoplasma spp., originally lyophilised and then reconstituted and stored at -70 degrees C, were viable after 16 years. Viable organisms were found, with little or no reduction in titre, in all of seven broth cultures of Ureaplasma urealyticum, comprising six serotypes, after storage for 6-10 years at -70 degrees C, but five of 18 broth cultures of other human and animal ureaplasmas stored likewise were not viable after 13-14 years and in a further seven of them the titre of viable organisms had diminished greater than or equal to 10(4)-fold.
Topics: Animals; Bacteriological Techniques; Cell Division; Cryopreservation; Culture Media; Freeze Drying; Humans; Mycoplasma; Time Factors; Ureaplasma
PubMed: 2179556
DOI: 10.1099/00222615-31-3-203 -
Biochemical and Biophysical Research... Apr 1997Mycoplasma fermentans has unique glycoglycerophospholipids (GGPLs: GGPL-I and GGPL-III). Previously, the structure of these lipids was determined as... (Comparative Study)
Comparative Study
Mycoplasma fermentans has unique glycoglycerophospholipids (GGPLs: GGPL-I and GGPL-III). Previously, the structure of these lipids was determined as phosphocholine-6'-alpha-glucopyranosyl-(1'-3)-1, 2-diacyl-glycerol (GGPL-I) and 1"-phosphocholine-2"-aminodihydroxypropane-3"-phospho-6'-alph++ + a- glucopyranosyl-(1'-3)-1, 2-diacyl-glycerol (GGPL-III). Thin-layer chromatography (TLC) immunostaining showed that the GGPLs were main lipid-antigens of the M. fermentans species. Anti-M. fermentans serum stained mainly the GGPLs, but the other anti-mycoplasma sera (anti-M. arginini, anti-M. hyorhinis, anti-M. pneumonia, anti-M. primatum, and anti-Acholeplasma laidlawii, anti-M. hominis, anti-M. orale, and M. salivarium) stained neither GGPL-I nor GGPL-III. The TLC analysis of glycolipids and phospholipids of various human related mycoplasmas showed clearly that GGPLs are specifically expressed in M. fermentans species. GGPL-I and GGPL-III ranged from 1.6 to 28% and from an undetectable level to 35% of total phospholipids, respectively. Although there was heterogeneity among the amounts of GGPL-I or GGPL-III of M. fermentans strains, all of the M. fermentans strains had GGPL-I and/or GGPL-III. These observations showed that GGPL structures are species-specific immunodeterminants of M. fermentans. The fact that the GGPLs are main phospholipid components of the M. fermentans species means the M. fermentans has a unique choline metabolic pathway. This observation may raise phylogenetic interest.
Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Glycolipids; Humans; Molecular Structure; Mycoplasma; Mycoplasma fermentans; Phosphorylcholine; Rabbits; Species Specificity
PubMed: 9168906
DOI: 10.1006/bbrc.1997.6443 -
The American Journal of Tropical... Nov 1989The newly recognized pathogenic virus-like infectious agent (VLIA), originally reported in patients with AIDS but also known to be pathogenic in previously healthy...
The newly recognized pathogenic virus-like infectious agent (VLIA), originally reported in patients with AIDS but also known to be pathogenic in previously healthy non-AIDS patients and in non-human primates, was cultured in cell-free conditions using a modified SP-4 medium and classified as a member of the order Mycoplasmatales, class Mollicutes. The infectious microorganism is tentatively referred to as Mycoplasma incognitus. M. incognitus has the unique biochemical properties of utilizing glucose both aerobically and anaerobically, as well as having the ability to metabolize arginine. Among all known human mycoplasmas, these specific biochemical characteristics were found previously only in a rarely isolated species, M. fermentans. In comparison with M. fermentans, M. incognitus appears to be even more fastidious in cultivation requirements and fails to grow in all tested mycoplasma media other than modified SP-4 medium. In addition, M. incognitus grows much more slowly, has a smaller spherical particle size and occasional filamentous morphology, and forms only irregular and very small colonies with diffuse edges on agar plates. Antigenic analysis using polyclonal and monoclonal antibodies and DNA analysis of sequence homology and restriction enzyme mappings in M. incognitus, M. orale, M. hyorhinis, M. hominis, M. pneumoniae, M. fermentans, M. arginini, M. genitalium, M. salivarium, Ureaplasma urealyticum, and Acholeplasma laidlawii revealed that M. incognitus is distinct from other mycoplasmas, but is most closely related to M. fermentans.
Topics: Animals; Antigens, Bacterial; Arginine; Culture Media; DNA, Bacterial; Genes, Bacterial; Glucose; Humans; Mice; Mice, Inbred BALB C; Microscopy, Electron; Mycoplasma; Mycoplasma Infections; RNA, Ribosomal; Sequence Homology, Nucleic Acid
PubMed: 2817215
DOI: 10.4269/ajtmh.1989.41.586 -
Journal of Bacteriology Jul 1981Acetate kinase activity was assayed in 13 mycoplasmas. Nine species exhibited the enzymic activity in the direction of either synthesis of acetylphosphate or adenosine... (Comparative Study)
Comparative Study
Acetate kinase activity was assayed in 13 mycoplasmas. Nine species exhibited the enzymic activity in the direction of either synthesis of acetylphosphate or adenosine triphosphate. On the other hand Mycoplasma orale, Mycoplasma arthritidis, Ureaplasma urealyticum (10 serotypes), and two strains of Anaeroplasma species exhibited only minimal levels of the enzymic activity. In these four species, the enzyme does not seem to play a key role in adenosine triphosphate formation.
Topics: Acetate Kinase; Acholeplasma; Adenosine Triphosphate; Mycoplasma; Phosphotransferases; Species Specificity; Ureaplasma
PubMed: 6263869
DOI: 10.1128/jb.147.1.271-273.1981 -
Applied and Environmental Microbiology Mar 1994A fast and simple method to detect mycoplasmal contamination in simulated samples of animal sera by using a PCR was developed. The following five mycoplasma species that...
A fast and simple method to detect mycoplasmal contamination in simulated samples of animal sera by using a PCR was developed. The following five mycoplasma species that are major cell culture contaminants belonging to the class Mollicutes were investigated: Mycoplasma arginini, Acholeplasma laidlawii, Mycoplasma hyorhinis, Mycoplasma orale, and Mycoplasma fermentans. After a concentration step involving seeded sera, genus-specific primers were used to amplify a 717-bp DNA fragment within the 16S rRNA gene of mycoplasmas. In a second step, the universal PCR was followed by amplification of variable regions of the 16S rRNA gene by using species-specific primers, which allowed identification of contaminant mycoplasmas. With this method, 10 fg of purified DNA and 1 to 10 color-changing units of mycoplasmas could be detected. Since the sensitivity of the assay was increased 10-fold when the amplification products were hybridized with an internal mycoplasma-specific 32P-labelled oligonucleotide probe, a detection limit of 1 to 10 genome copies per PCR sample was obtained. This highly sensitive, specific, and simple assay may be a useful alternative to methods currently used to detect mycoplasmas in animal sera.
Topics: Animals; Base Sequence; Blood Specimen Collection; Cattle; Horses; Molecular Sequence Data; Polymerase Chain Reaction; Sensitivity and Specificity; Species Specificity; Tenericutes; Time Factors
PubMed: 8161186
DOI: 10.1128/aem.60.3.953-959.1994