Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Technical Tipslllllll Detection by Studies of highly conserved 16S rRNA or mouse leukemia cell line L1210. sequences of the class Mollicutes have The number of different RFLP pro- PCR and improved our understanding of their files obtained after digestion of the 1.5- origin and phylogenetic rela- kb 16S rRNA gene fragments from 11 Differentiation tionships.ll-81 PCR amplification of ap- mollicutes species and C. sordelli were: by Restriction proximately 1-kb DNA sequences of EcoRI-HindIII, seven (Fig. 2B); HaeIII- 16S rRNA genes from several species of HindIII, ten (Fig. 2C); AluI, twelve (Fig. Fragment Length the genus Mycoplasma, a member of 2D); and HindlII, five (data not Polymorphism of this class, has been reported for the shown). Because multiple isolates of detection and identification of several the various species have not been Acholeplasma, mycoplasmas, including the human tested, we do not know how well the parasites Mycoplasma salivarium, M. RFLP profiles are conserved within Spiroplasma, orale, M. genitalium, and M. hominis, tg) single species. However, such studies Mycoplasma, and In this report, we describe the use will be undertaken in the near future. of two universal PCR primer pairs (Fig. Upstream primer A, from the 5' Ureaplasma, 1) for the amplification of 1.5-kb and terminus portion of E. coli 16S rRNA, 1.1-kb 16S rRNA genes from various with two nonhomologous nucleotides Based upon 16S mollicutes. Restriction fragment length at its 3'terminus, prevented the rRNA Genes polymorphism (RFLP) typing of the amplification of the E. coli 16S rRNA amplified 1.5-kb 16S rRNA genes was gene. Because there was no amplifica- used to establish mollicutes species- tion of the DNA of insect or mam- Sujun Dengl, 4, Chuji Hiruki, 1 specific RFLP profiles. malian cell lines, the method may be appropriate for the detection of mol- Janet A. Robertson, 2 and RESULTS AND DISCUSSION licutes in their arthropod vectors and Gerald W. Stemke 3 DNA fragments of 1.5 kb and 1.1 kb mollicutes present in humans and were amplified from genomic DNAs of animals. This would be especially use- 1Department of Plant Science, four Acholeplasma species, three ful for detecting microorganisms that 2Department of Medical Microbiology Spiroplasma species, nine Mycoplasma presently are difficult or impossible to and Infectious Diseases, 3Department culture. Furthermore, the amplified of Microbiology, University of Alberta, species, and Ureaplasma urealyticum, Edmonton, Alberta, Canada T6G 2P5 and the gram-positive bacterium, DNA can be sequenced directly and Clostridium sordelli (Table 1). Plasmid these sequences can be used to estab- pMf6 with the full-length 16S rRNA lish phylogenetic relationships. Theo- gene insert of M. flocculare was used as retically, PCR is capable of detecting positive control. The amplified DNA single DNA or RNA molecules, even in fragments were all of the predicted the presence of large amounts of ex- length between the two primer pairs traneous DNA, allowing a single mol- and all hybridized with the cloned 16S licutes cell to be detected without cul- rRNA gene of M. flocculare. No detec- ture. table amplification occurred with DNA We emphasize that our results dif- templates of Escherichia coli, P. vulgaris, fer from those reported recently by Providencia spp., insect species Macro- Blanchard et al.. ~9) Their primer pair steles fascifrons and Aphrodes bicinctus, was much more selective than ours, human leukemia cell line CCRT-CEM, and their method had the advantage 1082 PRIMER B' 1103 1493 PRIMER B 1515 TACAACC CAATI'CAG GGC GTI~ TCAGCATrGTTC CATAGGGATG G 0 5'-I 16S rRNA GENE I- 3' AGAGTITGATCCTG G CTCAGGA 4present address: Animal Research 8 PRIMER A 29 Centre, Agriculture Canada, Ottawa, FIGURE 1 Location and composition of deoxyoligonucleotide primers complementary to the Ontario, Canada K1A 0C6. 5' and 3' termini of mollicutes 16S rRNA genes. 202 PCR Methods and Applications 1:202-2049 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/92 $3.00 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 11|1111 Technical Tips TABLE 1 Summary of Amplification Results of Small Subunit Ribosomal RNA probe did hybridize with the amplified Genes gene fragment from C. sordelli. We have not examined the ability of this Strain or assay to identify mollicutes in the Species Host ATCC# 1.5 kb 1.1 kb presence of Gram-positive bacteria. Genus Acholeplasma The 165 rRNA genes of many Gram- A. axanthum plants 25176 + 4- positive bacteria besides C. sordelli have A. granularurn animals NIAID + + similar conserved sequences recog- A. laidlawii animals B 4- 4- nizable by our primers and thus would A. modicum animals 10134 4- 4- be amplified. However, it is not likely that they would have RFLP profiles Genus Spiroplasma identical with those of the mollicutes. S. cirri plants, insects 27556 4- + Gram-positive bacteria would probably S. mellifentm insects 33219 + + Flower spiroplasma plants 43260 + + not be frequently found as cell culture contaminants because of the wide- Genus Mycoplasma spread inclusion of penicillin in the M. arginini animals 23838 + 4. media. If present, they could be M. fennentans humans incognitus + + detected by the Gram stain or by classi- M. flocculare swine 27399 4- + cal bacteriological culturing methods. M. hominis humans 14027 + + We tested four of the five most M. hyopneumoniae swine 25095 + nt common rnycoplasmal contaminants M. hyorhinis swine 17981 + nt of cell cultures, namely, A. laidlawii, M. M. pinm~ animals F-38 + + arginini, M. fermentans, and M. hyor- M. pneurnoniae humans 15531 + nt M. salivarium humans 23064 + nt hinis. The 165 rRNA genes of all were amplified. The fifth common con- U. urealyticum animals T960 + § taminant, M. orale, was not examined. However, as the PCR primers are com- Clostridium sordelli humans 9714 + + plementary to the 5' and 3' termini Escherichia coil mammals K12 - - of the published 165 rRNA sequence of Proteus vulgaris various - - this microorganism (GenBank se- Providencia spp. mammals lab strain - - quence accession no. M24659), we ex- pect it to be amplified by this proce- Insects Macrosteles fascifrons dure. Because eukaryotic ribosomal Aphrodes bicinctus DNA is not amplified, PCR amplifica- tion with these primers should also be Cell lines applicable to Spiroplasma- or Achole- Human leukemia CCRF-CEM CCL 119 plasma-infected arthropods or myco- Mouse leukemia L1210 CCL 219 plasma-like organisms (MLOs) in insect vectors. This would be especially useful Plant tissues because few of these microorganisms Catharanthus roseusa 4- 4- have been isolated or characterized (nt) Not tested; (4-) amplification test positive; (-) amplification test negative. and their roles in the ecosystem are aChloroplast 165 rRNA genes were amplified with the universal primers. poorly understood. Noncharacterized mollicutes species can be partly identi- that Gram-positive bacterial DNA was amplified 165 rRNA genes from M. fied by comparing the established 165 not amplified. However, their up- hyorhinis and M. hyopneumoniae, and C. rRNA gene-RFLP profiles or their se- stream primer was, in fact, largely sordelli (data not shown). HaelII- quence data. We do not recommend specific only to the "hominis" cluster of HindlII RFLP typing differentiated the using these primers to amplify 165 mycoplasmas, (4) and thus U. urea- amplified 165 rRNA genes from C. sor- rRNA genes of MLOs directly from lyticum and M. pneumoniae, members of delli and all mollicutes tested (Fig. 2C) chloroplast-containing plant tissue be- the "pneumoniae" cluster, and the two except A. modicum, A. axanthum, and cause plant chloroplast 165 rRNA genes species of greatest importance in hu- M. arginini; these had identical RFLP apparently were amplified (Table 1). man disease, were not amplified. profiles (Fig. 2C, lanes 11, 14, 10). The The results of this investigation EcoRI-HindllI RFLP profiles differ- amplified 165 rRNA genes from all 11 suggest that a PCR-RFLP kit should be entiated the amplified 165 rRNA genes mollicutes species and C. sordelli were capable of detecting not only mol- from M. hyopneumoniae and M. distinguishable by AluI RFLP typing licutes in cell culture or its com- pneumoniae, and C. sordelli (Fig. 2B, (Fig. 2D). These results indicate that ponents, but also mollicutes species as- lanes 16, 6, 4), but not those genes HaelII-HindIII and AluI will distinguish sociated with human, animal, and in- from the other nine mollicutes species. 165 rRNA genes of many mollicutes. sect diseases, and of predicting the HindlI1 RFLP typing differentiated the The mycoplasma 165 rRNA gene phylogenetic relationships of these fas- PCR Methods and Applications 203 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Technical TipslllllllEE cinating microorganisms. ACKNOWLEDGMENTS We are grateful to T. Tribe and Gina Figueiredo for technical assistance. This research was supported by Operat- ing Grants A3843 and OGP0007320 from the Natural Sciences and Engineering Research Council of Cana- da to C.H. and G.W.S., respectively, and Operating Grant 7759 from the Medical Research Council of Canada to J.A.R. REFERENCES 1. Frydenberg, J. and C. Christiansen. 1985. The sequence of 16S rRNA from Mycoplasma strain PG50. DNA 4: 127-137. 2. Maniloff, J. 1983. Evolution of wall- less prokaryotes. Annu. Rev. Microbiol. 37: 477-499. 3. Taschke, C., K. Ruland, and R. Herrmann. 1987. Nucleotide se- quence of the 16S rRNA of Myco- plasma hyopneumoniae. Nucleic Acids Res. 15:3918 4. Weisburg, W.G., J.G. Tully, D.L. Rose, J.P. Petzel, H. Oyaizu, D. Yang, L. Mandelco, J. Sechrest, T.G. Lawrence, J. Van Etten, J. Maniloff, and C.R. Woese. 1989. A phylogenetic analysis of the mycoplasmas: Basis for their classification. ]. Bacteriol. 171: 6455- 6467. 5. Woese, C.R. 1987. Bacterial evolu- tion.