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J Vet Diagn Invest 9:186-190 (1997)
Polymerase chain reaction and restriction endonuclease
digestion for selected members of the “ Mycoplasma mycoides cluster”
and Mycoplasma putrefaciens
Jose L. Rodriguez, Richard W. Ermel, Thomas P. Kenny,
Dale L. Brooks, Al J. DaMassa
Abstract. A specific diagnostic method using the polymerase chain reaction, together with restriction en- donuclease digestion patterns, was developed for members of the “ Mycoplasma mycoides cluster” that normally occur in the United States (i.e., Mycoplasma mycoides subsp. mycoides Large Colony and Mycoplasma capri- colum subsp. capricolum in addition to “cluster” mycoplasma, bovine serogroup 7, and Mycoplasma putrefaciens. The digestion of “cluster” polymerase chain reaction DNA (1,225 bp) amplification products with restriction enzymes AseI and SspI gave mycoplasma species-specific patterns for all strains of M. mycoides subsp. mycoides Large Colony, M. capricolum subsp. capricolum, and bovine group 7 tested. Moreover, we found a nonspecific amplification product for M. putrefaciens that occurred with the oligonucleotide primers used for the “ M. mycoides cluster” reaction. However, the restriction endonuclease digestion patterns observed with the restriction enzymes AluI, AseI, and SspI for M. putrefaciens were different than the digestion patterns obtained for the other “cluster” mycoplasmas. This report confirms the usefulness of polymerase chain reaction DNA ampli- fication allied with restriction enzyme digestion profile analysis for the rapid and specific identification of mycoplasmas belonging to the “ M. mycoides cluster” and M. putrefaciens.
Mycoplasma infections in ruminants are frequently (^) one serious outbreak/ For the purpose of this report, observed, particularly in goats. However, it is extreme- (^) the “ M. mycoides cluster” shall infer only MmLC, MC, ly important to distinguish between mycoplasmas and IM. sp. serogroup 7. Other members of the “clus- causing severe and contagious diseases with serious ter” were not analyzed due to their “exotic” status in economic consequences and mycoplasmas that may (^) the United States. cause sporadic disease with minor economic impact. In addition to the basic questions of taxonomy with- For example, worldwide, the most important myco- in the “cluster,” differential diagnostic tests are often plasmas economically are the related ruminant patho- difficult to develop due to shared common antigens gens classified as the “ Mycoplasma mycoides cluster.” among these mycoplasma.^10 Cross-reactive antibodies This “cluster” 4 consists of six mycoplasma species: can impede the identification of the pathogenic agents Mycoplasma mycoides subsp. mycoides Small Colony thereby complicating the interpretation of serological (MmSC), Mycoplasma mycoides subsp. mycoides Large tests.^6 Moreover, biochemical differentiation may be Colony (MmLC), Mycoplasma mycoides subsp. capri time-consuming and ambiguous. Recently, DNA (Mmc), Mycoplasma capricolum subsp. capricolum probes have been used to develop more specific di- (MC), Mycoplasma capricolum subsp. capripneumon- agnostic procedures. 7,14,15^ These tests may be compli- iae (Mcc), and an unspeciated bovine mycoplasma des- cated by nonspecific binding of probes to nontarget ignated “bovine serogroup 7.” areas with poor reproducibility and difficult interpre- In the United States, the “cluster” mycoplasmas tation. Similarly, the comparison of restriction endo- causing disease, principally in goats, are MmLC, which nuclease digestion patterns is an additional molecular is widespread, and MC, which is reported sporadically. classification criterion, but this procedure often re- Moreover, Mycoplasma putrefaciens (Mp) is often re- quires sophisticated digitizing scanning to remove sub- ported in disease processes and Mycoplasma agalac- jectivity and obtain precise data comparisons.3,12,13, tiae has been described on three occasions, including Recently, tests based on the polymerase chain re- action (PCR) have proven to be highly specific and inexpensive for the identification of MmSC.2,8^ Thus, From the Department of Pathology, School of Veterinary Medi- tine, University of Las Palmas de Gran Canaria, Spain (Rodriguez),
the objective of this study was to develop a rapid and Department of Medicine, School of Veterinary Medicine (Ermel, specific diagnostic method using the PCR for DNA Brooks, DaMassa), and Rheumatology Division, School of Medicine amplification of the “ M. mycoides^ cluster.” This re- (Kenny), University of California, Davis, CA 95616. action was coupled with restriction endonuclease di- Received for publication April 13, 1995. gestion patterns to distinguish the ruminant myco- 186
PCR for “Mycoplasma mycoides cluster” and Mycoplasma putrefaciens (^) 187 Table 1. Mycoplasmas used in this study.
plasma “cluster” pathogens commonly described in the United States.
Materials and methods Mycoplasma. Table 1 lists the mycoplasma species and sources included in this study along with three recently de- scribed caprine mycoplasma species: Mycoplasma auris, My- coplasma cottewii, and Mycoplasma yeatsii.^5 Mycoplasma media. Liquid and solid mycoplasma me- dium “B,” as described elsewhere, were used in this study.^9 DNA extraction. Template DNA was extracted by two methods. In the first method, a proteinase K digestion was followed by a phenol-chloroform extraction (1 ml of broth culture with about 10^7 -10^9 colony-forming units of the my- coplasma) and centrifuged for 10 minutes at 14,000 x g. The pellet was resuspended in 600 μl of TE buffera^ (10 mM Tris- HCl, pH 8.0, 1 mM EDTA) containing 0.5% sodium dodecyl sulphate to which was added 3 μl of proteinase Ka^ (20 mg/ ml). After incubation for 3 hr at 37 C, the lysate was extracted twice with 500 μl of phenol-chloroform isoamyl alcohola^ (25: 24:1). The DNA was precipitated with 50 μl of 3 M sodium acetatea^ and 1 ml of cold 95% ethanol and left overnight at -20 C. After 30 min centrifugation, the DNA was washed with 70% ethanol, dried, and resuspended in 100 μl of deion- ized water. One microliter of extracted DNA template was used in the PCR amplifications. The second DNA extraction method was with “Genere- leaser,”b^ which is a proprietary reagent that releases DNA
from whole blood, cell cultures, and bacterial colonies, etc. One microliter of broth culture and 5 μl of “Genereleaser” were mixed, and the extractions were made within 10 min according to the manufacturer’s instructions. Oligonucleotide primer design. (^) Primers were designed from the published nucleic acid sequences of the six “M. mycoides cluster” species that are complementary to the gene probe CAP-21.^15 Briefly, two PCR primers were designed for the mycoplasmas belonging to the “cluster” (Table 2). These primers (1 and 2) were selected in DNA regions where com- plete homology was present between the six sequences, giving an amplified DNA segment of about 403 bp, depending on the “cluster” species involved. In addition, two sets of 5' and 3' oligonucleotide primers were designed to give specific am- plification products for MmLC and MC (Table 2). The prim- ers 3, 4, and 7 were specific for MmLC, while primers 5 and 6 were specific for MC. These primers were selected from regions in which there were differences between the “cluster” species. One microliter of extracted DNA and 10 μl of each primerc (2 μM concentration) were added to 30 μl of the PCR mixture d that consisted of 5 μl thermophilic buffer 10 x (500 mM KCl, 100 mM Tris-HCl, 1% Triton X-100) 3 μl 25 mM MgCl 2 , 2 μl 1.25 mM (each) dATP, dGTP, dTTP, and dCTP, and 1.5 units Taq polymerase. d^ The mixture was amplified in a thermal cycler for 30-40 cycles with each cycle consisting of melting at 94 C for 1 min, annealing at 50-60 C for 1 min, and extension at 72 C for 2 min. The concentrations of MgCl, and the 5' and 3' primers were varied to improve the spec-
PCR for “Mycoplasma mycoides cluster” and Mycoplasma putrefaciens 189
Figure 2. DNA amplification products of Mycoplasma putrefa- ciens (Mp) strains KS1, GM1, GM499, GM1032, and “Mycoplasma mycoides cluster” on an agarose gel stained with ethidium bromide. Lanes are: 1, negative control without template DNA, 2, Mp tem- plate DNA without digestion with restriction enzymes; 3, negative control (DNA from blood of noninfected goat); 4, MmLC template DNA digested with enzyme AluI (615 and 360 bp); 5, 6, 7, and 8, Mp template DNA strains KS1, GM1, GM499, and GM1032, re- spectively, digested with AluI; 9, 10, 11, and 12, Mp template DNA strains KS1, GM1, GM499, and GM1031, respectively, digested with AseI; 13, 14, 15, and 16, Mp template DNA strains KS1, GM1, GM499, and GM1031, respectively, digested with SspI.
bands (Fig. 2). This digestion pattern was different than the AluI pattern observed for MmLC, MC, and bovine serogroup 7. The restriction pattern with enzyme AseI was characterized by three bands of about 750, 350, and 150 bp. With enzyme SspI, the size of the bands were similar to those detected with MC (380, 330, and 180 bp).
Discussion The existence of both Small and Large Colony types of M. mycoides subsp. mycoides has presented prob- lems with serologic testing for many years. 6, In coun- tries such as the USA, which is reported to be free of the SC type of the species, specific diagnostic tests are required to differentiate it from the prevalent caprine LC type.^7 Likewise, the occurrence of MC and possibly bovine serogroup 7 makes the diagnostic testing and distinction of these “cluster” mycoplasmas very im- portant. The data in this study identify a set of fast, sensitive, and reliable tests for the differentiation be- tween all of the “cluster” mycoplasmas and Mps that have been described in the USA. Our initial trials, which were directed toward the development of a one-step MmLC- and MC-specific PCR assay, did not provide definitive and specific bands for a reliable diagnostic test. These results were most likely due to the high degree of sequence similarity between the different mycoplasmas within the “clus- ter” group. For example, the differences between the specific mycoplasma primers and the DNA of other “cluster” mycoplasmas were in two or three nucleo-
tides, which is not enough to establish a specific di- agnostic DNA amplification test. The poor specificity could also be due to the high proportion of adenine and thymidine in our primers that prevented the use of high annealing temperatures to improve the speci- ficity of the primers. In subsequent experiments, the use of the PCR assay followed by restriction endonuclease digestions gave specific results confirming the amplified DNA frag- ments as MmLC, MC, or bovine serogroup 7. The unique DNA amplification band of about 1,225 bp with primers 2 and 8 confirmed the specificity of these primers as previously reported.^14 Moreover, the clear multiple bands observed after digestion with the re- striction enzymes AseI and SspI for the reference strains of each mycoplasma confirmed the presence of species- specific internal restriction sites. Four strains of MmLC and six strains of MC showed bands similar to those produced by the reference strains. An extra band of about 170 bp was observed with the enzyme AseI in all of the MmLC strains and may indicate one extra restriction site around nucleotide 64 of the MmLC sequence. The PCR amplification with primers 2 and 8 followed by digestion with restriction enzymes AseI and SspI provided excellent differential identification for the USA strains within the “cluster.” Interestingly, the digestion patterns for strain GM of MC with enzyme SspI indicated some potential mi- nor sequence differences from the reference strain of the species. However, with the addition of enzyme AseI the digestion pattern allowed the classification of this strain as MC. The restriction patterns of MmSC with enzyme AsnI were different from the other “clus- ter” mycoplasmas. 2,15^ With our oligonucleotide prim- ers, enzymes AseI and AsnI cut the same nucleotides, producing restriction digestion fragments that were 750, 400, and 86 bp. Thus, this restriction digestion test can be used diagnostically to distinguish MmSC from all the other “cluster” mycoplasmas. The nonspecific reaction observed with Mp inter- feres with the specific diagnosis of other “cluster” my- coplasmas by PCR amplification followed by restric- tion enzyme digestion when only one enzyme is used. 2 However, the combination of three enzymes was di- agnostic for Mp, providing a restriction pattern that was different from the “cluster” mycoplasmas. Pres- ently, this series of tests will be very useful for the diagnostic identification of Mp. The apparent similarity between the PCR amplifi- cation products obtained with Mp and the other “clus- ter” mycoplasmas indicates a high degree of homology. Other investigators have sequenced 16S rRNAs from Mp and, based on sequence homology, have suggested that Mp should be included in the “ M. mycoides clus- ter. ”1,16^ Our results support this suggestion.
190 Rodriguez et
In summary, this report confirms the usefulness of the PCR and restriction endonuclease digestion for the rapid and specific identification of the ruminant my- coplasmas listed in Table 1. Our experiments have indicated that a set of tests including PCR DNA am- plifications followed by restriction endonuclease di- gestions can be used to make a diagnosis in less than 24 hours. This set of reliable, sensitive, and quickly performed diagnostic tests has great economic poten- tial and could be substituted for other time-consuming DNA techniques. 1,2,7,8, Studies with “exotic” strains of the “ M. mycoides cluster” from other countries should be conducted to further evaluate the specificity of these procedures.
Acknowledgement We would like to express our gratitude to the University Foundation of the University of Las Palmas de Gran Canaria for the grant provided to J. L. Rodriguez that allowed him to pursue these studies at the University of California, Davis.
Sources and manufacturers Sigma Chemical Company, St. Louis, MO. Bioventure Inc., Murfreesboro, TN. The Midland Certified Reagent Company, Midland, TX. Promega, Madison, WI. Pharmacia Biotech Inc., Piscataway, NJ.
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- Bergemann AD, Whitley JC, Finch LR: 1990, Taxonomic 17. significance of differences in DNA methylation within the My- coplasma mycoides cluster detected with restriction endonu- cleases MboI and DpnI. Lett Appl Microbiol 11:48-51.
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