Detection of mycoplasma contaminations by the polymerase chain reaction
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Agarose gel electrophoresis
genomic DNA
Mycoplasmas are very small bacteria lacking cell walls that belong to various genera within the class Mollicutes, and also the smallest organisms that can live independently. They are able to cause serious and chronic disease because of some unique characteristics. Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economical losses within the poultry industry. The aim of the present study was to investigate the prevalence of M. gallisepticum in poultry by PCR of mgc2 and 16 s rRNA. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the 16S rRNA and mgc2 genes present in MG. For serological screaming test, we selected 26 farms and took blood samples for RSAT assay. For 16S rRNA and mgc2 PCR assay, we took 109 samples from 10 rapid slide agglutination test (RSAT) positive farms, including: lung, air sacs and tracheal swabs. The 16S rRNA and mgc2 PCR diagnostic primers are specific for MG in tests of all avian Mycoplasmas or bacteria present in the chicken trachea and are sensitive enough to readily detect MG in tracheal swabs from field outbreaks. 530 bp and 300 bp PCR products on electrophoresis gel appeared respectively with 16S rRNA and mgc2 PCR diagnostic primers, specific for MG. The test was successfully applied in vivo for detection of MG in clinical samples.
Mycoplasmataceae
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The taxonomy of canine Mollicutes is described, based on phylogenetic analysis of 16S rRNA gene and 16S/23S rRNA intergenic spacer (IGS) region sequences. The nucleotide sequences of the 16S rRNA gene of two untyped mycoplasmas and the IGS region of 11 Mycoplasma species were determined and used for phylogenetic analysis. The two untyped Mycoplasma strains, HRC 689 and VJC 358, were found to be distinct from all known canine mycoplasmas and all published mycoplasma 16S rRNA gene sequences.
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Infections with Mollicutes species (such as Mycoplasma, Acholeplasma, and Ureaplasma) can induce a variety of problems in living organisms and laboratory cell cultures. Therefore, it is necessary to establish a routine diagnostic protocol for Mycoplasma infection in order to ensure reliable research results, as well as the safety of commercial biological products. For that purpose a novel PCR-based procedure using specific designed primers complementary to 16S rRNA genome region of mollicute species was evaluated. PCR was optimized and sensitivity and specificity was evaluated by defined cell count concentrations (2-31250 CFU/ml) of different strains of Mycoplasma, Acholeplasma and Ureaplasma. Amplicon (272 bp) was cloned by PCR-cloning and sequenced by dideoxy chain termination. PCR, was found to be able to detect 10 copies of mollicute target DNA. No cross-reactivity with genomic DNA of non-mollicute bacteria or human cell lines was observed. Forty seven human and animal cell lines were evaluated for mollicute contamination. Twenty five cell lines (53%) were correctly identified as contaminated by this molecular approach. The results of this study demonstrated that this PCR-based method is not only fast and reproducible, but also highly sensitive and specific for detecting contaminant mycoplasmas in cell cultures.
Ureaplasma
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Agarose gel electrophoresis
genomic DNA
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Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Infections with Mycoplasma species can cause a variety of problems in living organisms and in vitro cell cultures. In this study, we investigated the usefulness of a genus-specific consensus PCR analysis method to detect Mycoplasma species. The developed consensus primer pairs MycoF and MycoR were designed specifically to amplify the 16S ribosomal RNA gene (rRNA) of Mycoplasma species by the optimized PCR system. The developed consensus PCR system effectively amplified 215 bp of Mycoplasma genus-specific region of 16S rRNA. In conclusion, we recommend this consensus PCR for monitoring Mycoplasma species in animals, human and cell culture system.
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The identification of cell cultures contaminated with organisms from the class Mollicutes has led us to examine the effectiveness of polymerase chain reaction (PCR) for detecting these organisms in genomic DNA. We developed a previously identified nested PCR primer set and compared its ability to detect Mycoplasma with that of a commercially available PCR kit for detecting Mycoplasma. We found that although the commercial system detected and identified a few of the most common Mycoplasma species, the primer set (GPO-1, GPO-2, MGSO) detected the presence of all the common Mycoplasma species and many of the rare mycoplasma species previously encountered in tissue culture.
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genomic DNA
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Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Infections with Mycoplasma species can cause a variety of problems in living organisms and in vitro cell cultures. In this study, we investigated the usefulness of a genus-specific consensus PCR analysis method to detect Mycoplasma species. The developed consensus primer pairs MycoF and MycoR were designed specifi-cally to amplify the 16S ribosomal RNA gene (rRNA) of Mycoplasma species by the optimized PCR system. The developed consensus PCR system effectively amplified 215 bp of Mycoplasma ge-nus- specific region of 16S rRNA. In conclusion, we recommend this consensus PCR for monitoring Mycoplasma species in animals, human and cell culture system.
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23S ribosomal RNA
Mycoplasmataceae
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Mycoplasma hominis is a heterogeneous species with DNA-DNA hybridization values ranging from 51 to 100%. We report here the sequencing of the 16S rRNA gene of a strain (183) that greatly differs from the type strain (PG21) of this species. Comparison of 16S rDNA sequences from these two strains showed limited differences, indicating that the two strains belong to the same rRNA species complex. Using these nucleotide sequence data, we established a rapid method for the detection of M. hominis by using polymerase chain reaction. This method was shown to be sensitive and specific when tested with reference strains and clinical isolates.
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Journal Article Human ureaplasmas show diverse genome sizes by pulsed-field electrophoresis Get access Janet A. Robertson, Janet A. Robertson Search for other works by this author on: Oxford Academic PubMed Google Scholar Louise E. Pyle, Louise E. Pyle 1Russell Grimwade School of Biochemistry, University of MelbourneParkville, Victoria 3052, Austraiia Search for other works by this author on: Oxford Academic PubMed Google Scholar Gerald W. Stemke, Gerald W. Stemke 2Department of Microbiology, University of AlbertaEdmonton, Alberta, Canada T6G 2H7 Search for other works by this author on: Oxford Academic PubMed Google Scholar Lloyd R. Finch Lloyd R. Finch * 1Russell Grimwade School of Biochemistry, University of MelbourneParkville, Victoria 3052, Austraiia *To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 18, Issue 6, 25 March 1990, Pages 1451–1455, https://doi.org/10.1093/nar/18.6.1451 Published: 25 March 1990 Article history Received: 12 December 1989 Revision received: 20 February 1990 Accepted: 20 February 1990 Published: 25 March 1990
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