CA1339547C - Detection of mycoplasma by dna hybridization - Google Patents
Detection of mycoplasma by dna hybridizationInfo
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- CA1339547C CA1339547C CA000512288A CA512288A CA1339547C CA 1339547 C CA1339547 C CA 1339547C CA 000512288 A CA000512288 A CA 000512288A CA 512288 A CA512288 A CA 512288A CA 1339547 C CA1339547 C CA 1339547C
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
Biological probes useful for detecting mycoplasmas or prokary-otes in general, or specific mycoplasma and eubacterial species are derived from the ribosomal RNA gene by selecting particular nucleotide sequences common to the class of organisms being detected.
Description
1~39~7 DETECTION OF MYCOPLASMA BY DNA HYBRIDIZATION
This invention was made with Government support under Grant No. AVAM 14096-01 with the National Institutes of Health and the University of California. The Govemment has certain rights in this inven-tion.
~ield of the Invention This invention relates generally to the ~leld of Biology, and more particularly to the fields of Biomedicine, 8iochemistry and Molecular Biology.
Back~round and Summary of the Invention Mycoplasmas are a group of pathogenic microor~anisms of the Class Mollicutes characterized by having a small size and lacking a cell wall. These microol~anisms are among the FnlAllest-known organisms capable of a free living existence, and are important pathogens in man, -2- 1~39547 plants and animals. For example, atypical pneumonia and non-gonococcal urethritis are common mycoplasma infections in man. Mycoplasmas have also been associated with rheumatoid arthritis, spontaneous abortion, infer-tility and other genital tract diseases, and certain autoimmune disease states. Moreover, mycoplasmas are common contaminants in cell cultures. In biological research, mycoplasma contamination of tissue culture is a serious problem which demands constant monitoring.
Not surprisingly, these organisms are extremely fastidious and at present there are no cost-effective specific diagnostic procedures to determine the presence of mycoplasma infections. The most commonly-employed detection methods for mycoplasmas in clinical samples are serological and cultural. The serological methods are subject to false positives and the cultural methods are costly, time consuming and tedious.
Many of the biochemical techniques in current usage for detection of microbial contaminants in cell cultures do not specifically detect myco-plasmas but rather indicate the presence of any prokaryote or simple eukaryote such as yeast and fungi, and some may even detect viruses. Such a test is advantageous if one is interested only in the knowledge that a microbial agent is present, but if one is searching for a suspected eti~
logical agent of an animal or human disease it is obviously necess&ry to classify the agent as fully as possible.
Further, the above proced~l,es are hampered by special prob-lems. ~or example, there are apparently "non-cultivable" mycopl~smas which are not detected by conventional culture methods. In addition, in the case of immunofluorescence tests more than one antibody might be re -3- 1339~547 quired to identify the particular organism since more than nine different mycoplasma species are common tissue culture cont~min~nts. Also, DNA
stains are not necessarily mycoplasma-specific.
Therefore, a simple, sensitive, specific, cost-effective, snd rapid mycopl~sma detection system has been a desideratum in the fields of diagnostic medicine and biological research.
The use of nucleotide sequence homology snd nucleic acid hybridization kinetics has become a widely-employe~d technique for detect-ing various organisms in cells and cell cultures. However, prior to this invention reliable and specific DNA probes have not been Qvailable for mycoplasma detection.
The present invention proceeds by the use of specific myco-pl~sma ribosomal RNA gene fragments which are lsbeled or tsgged by a variety of techniques, such as rsdioisotope l~beling, biotin labeling, PEI-peroxidase conjugates, or nuorescent antibody tagging ELISA methods, for the specific and sensitive detection of mycoplasmas in clinical specimens, cells or cell cultures by DNA or RNA hybridization.
In one sspect of the invention, a DNA sequence from the 16S
RNA gene of mycoplasma is provided, which includes a nucleotide sequence selected from the group consisting of AACACGTATC, CGAATCAGCTATGTCG, GAGGTT AAC, ATCCGGATTTATT, TCTCAGTTCGGATTGA, AGGTGGTGCATGGTTG, TCCTGGCTCAGGAT, ATACATAGGT, AACTATGTGC, AA ~ l~ACAATG, snd TCTCGGGTCT, which code for mycoplasma ribosomal RNA (rRNA) where T represents thymine, G represcnt guanine, A represents adenine, C repre-4~ 1339a~7 sents cytosine and - indicates a nucleotide deletion within the sequence with respect to the comparable sequence in E. coli. These fragments differ significantly from the 16S RNA gene of E. ~? and thus form the basis for mycoplasma-specific probes which are constituted of labeled nucleotide sequences complementary to the above.
In another aspect of the invention, identified DNA sequences of a 16S RNA gene are provided which include nucleotide sequences selected from the group consisting of ACGGGTGAGT, TAATACCGCAT, TACGGGAGGCAGCAGT, GTGGGGAGCAAA, AGGATTAGATACCCT, CCGTAAACGAT, GAATTGACGGGG, CCCGCACAAG, GGTGGAGCATGT, TGTTGGGTTAAGTCCCGCAACGA, GGGATGACGT, ACGTGCTACAATG, CTAGTAATCG, TGTACACACCGCCCGTCA, AAGTCGTAACAAGGTA, and TGGATCACCTCCTT, which code for prokaryotic rRNA. These frag-ments represent regions within the 16S RNA gene that are identical for E.
coli and all mycoplasmas examined. Universal probes for all prokaryotes are constituted of labeled nucleotide sequences complementary to these fragments.
In general the invention comprises a method for determining the presence of a prokaryotic organism which contains a nucleic acid including a particular nucleotide sequence which is present in nucleic acids from prokaryotic organisms but absent in nucleic acids from eukaryotic organ-isms, which comprises contacting a medium which may contain a nucleic acid or nucleic acid fragment from s~id prokaryotic organism including said particular nucleotide ~quence with an oligonucleotide, including a nucleo-tide sequence complementary to said particular nucleotide ~equence, 1339~47 whereby said oligonucleotide hybridizes with any nucleic acid or nucleic acid fragment from said prokaryotic organism, including said particular nucleotide sequence which may be present in said medium, and detecting the presence of any nucleic acid or nucleic acid fragment hybridized with said oligonucleotide.
Other aspects of the invention c~nccll. the specific biological probes used for detecting mycoplasmss or prokaryotes in general in accord-ance with the above described proce~ and the identification and production of such probes.
The sole figure of the drawing shows a slot blot development illustrating the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention will now be described by detailing first the spe-cific steps involved in producing and using the biological probes of the present invention and then describing how particular nucleotide sequences useful in this invention are determined.
SYNTHESIS OF DEOXYOLIGONUCLEOTIDES
The 16 bp deoxyoligonucleotide, GCTTAGTCGATACAGC, which is complementary to one of the 16S mycoplasma RNA gene sequences listed above, was synthesized using the phosphotriester solid phase procedure described in M. H. C~ruthers et aL, Genetic Engineerin~, -6- 1339~47 Vol. 4, p. 1-17, Plenum Publishing Co. (1982).
Any of the other deoxyoligonucleotides previously mentioned or any other desired oligonucleotide can be similarly prepared. For example, instead of a DNA probe it may be desired to synthesize an RNA probe such as a recombinant SP6 vector transcript containing the sequence CGAAUCAGCUAUGUCG.
DNA-RNA or RNA-RNA hybridization to ribosomal RNA mole-cules amplifies the sensitivity of the detection several hundredfold above any DNA-DNA or RNA-DNA hybridization using probes against genomic DNA sequences, since the use of such probe which will detect multiple copies of ribosomal RNA per mycoplasma or eubacterial cell.
TESTING OF DEOXYOLIGONUCLEOTIDES
The above described deoxyoligonucleotide was 32P-labeled at the 5' end using the procedure in C. C. Richardson, Procedures in Nucleic Acid Research (Cantoni, G. L. and Davies, D. R., eds.), Vol. 2, pp. 815-828, Harper and Row, New York (1971). The resultin~ labeled deoxyoligonucleotide was then used as a myco-plasma-specific probe. Specificity for mycoplasma was demonstrated by means of slot blot hyL. i~ ation as described in M. Cunningham, Anal.
Biochem. 128:41~421 l1983~.
For this purpose a 1600 bp DNA fragment of Mycoplasma pneumoniae which hsd been cloned into pUC8 was used. The 1600 bp fragment contains the above described 16 base deoxyoligonucleotide. A genomic digest of E.
~, a representative prokasyotic eubacterium, was also produced by -7- 133~47 digestion with the enzyme Hindm. The digested E. coli DNA ~nd the 1600 bp M. pneumoniae DNA fragment were trsnsferred onto nitrocellulose filters according to the procedure in the J. J. Leary et sl., Proc. Natl.
Acad. Sci. U.S.A. 80:4045-4049 (19831 The nitroc~ filters containing the DNA fr~mPnt~ were baked for 2 hours at 80~C under reduced pressure and hybridized to the 32P-labeled deoxyoligonucleotide. Development of the resulting slot blots, shown in the drawing, revealed blots of increasing intensity for the M.
pneumoni~e DNA segment at 0.00034 ng, 0.0034 ng, 0.034 ng, 0.34 ng, and 3.4 ng (calculated for 16 bp) and no blots for the E. coli DNA segment at 0.00015 llg, 0.0015 ~Ig, 0.015 llg, 0.15 llg,and 1.5 llg indicating the sp~
cificity of the deoxyoligonucleotide probe for mycoplasma. The deoxy-oligonucleotide having the sequence GCTTAGTCGATACAGC thus is useful as a mycoplasma-specific probe which hybridizes with mycoplasmal DNA
but does not hybridize with DNA of other prokaryotic organisms. On the other hand, a deoxyoligonucleotide having the sequence TGCCCACTCA, for example, which is complementary to one of the prokaryotic coding gene sequences, is useful as a prokaryote-specific probe which hybridizes with prokaryotes but not with eukaryotes.
For the detection of mycoplasmas in infected cells the follow-ing procedure h~ ~een found ef~ective. The cells are trypsinized using 1-_ T75 tissue culture flasks with Trypsin EDTA (0.05% trypsin, 0.04~ EDTA
in PBS) for 2 minutes at 3~C. The trypsinized cells are resuspended in 1-2 ml of growth medium and spotted in a quantity of 50-100 Ill (1 x 105 -1 x 106 cells) onto a nitrocellulose filter wetted with 10 x SSC (1 x SSC:
~ -8- 1339~47 15 mM Na citrate, 150 mM NaCl, pH 7.4) using a Minifold I[ slot blot hybri-dization apparatus available from Schleicher and Schuell, Inc., Keene, N. H. The DNA samples applied to the slot blots are denatured with alkali (0.5 M NaOH, 1.5 M NaCl) for 5-lOminutes at room temperature and neu-tralized for 5-10 minutes at room temperature using 0.5 M Tris, pH 7.2 and 3.0 M NaCl. The filter is then washed with 2 x SSC for five minutes at room temperature and baked in a vacuum oven for 2 hours at 80~C. The filter is prehybridized for 2 hours at 65~C using a prehybridization buffer consisting of 0.5 mM EDTA, 5 mM Tris, pH 7.5, 5 x Denhardt, and 100 ~g/ml heat denatured her,in~ sperm DNA. Hybridization, using the probes of this invention in a concentration measured as 1-2 x 106 cpm of 32P-labeled deoxyoligonucleotide, specific sctivity >108 cpm/~l g in hybridi-zation buffer consisting of 10 mM Tris, pH 7.5,1 mM EDTA, 0.75 M NaCl, 1 x Denhardt, 0.5% SDS, 10% dextran sulfate and 100 llg/ml heat dena-tured herring sperm DNA, is carried out at 65~C for 16 hours. Following hybridization the filter is washed 2-4 hours at 65~C with 2 x SET, 0.2% SDS
(1 x SET:30 mM Tris, pH 8.0, 150 mM NaCl) and 1-2 hours at room tem-perature with 4 mM Tris base. The Blter is then dried and exposed on X-ray film using 1 or 2 Dupont Cronex intensifying scree1ls.
DETERMINATION OF PARTICULAR NVCLEOTIDE SEQUENCES
While the foregoing description of the present invention teaches how particular nucleotide seguences can be prepared and used, the broader scope of this invention may be re~li7ed by ~y~minirlg the technigues used for determining particular nucleotide sequences which are useful as myc~
1~39~54 7 g plasma-specific probes, probes specific for prokaryotes in general, probes specific for individual mycoplasma, ureaplasma, acholeplasma, and spir~
plasma species or probes specific for individual eubacterial species.
Such determination involves the following steps:
1. cloning the entire genome of ribosomal RNA of a particul~r species of mycoplasma into a bacteriophage or plasmid vector;
2. probing the resulting ribosomal RNA gene fragments with a non-mycoplasm~ prokaryotic ribosomal RNA operon;
3. characterizing the fragments which hybridize with said non-mycoplasma prokaryotic ribosomal RNA operon;
4. identifying mycoplasma~pecific fragments by differential hybridization as described in Gobel, U. and Stanbridge, E. J., Science, VoL
226, pp. 1211-12~3 (1984).
This invention was made with Government support under Grant No. AVAM 14096-01 with the National Institutes of Health and the University of California. The Govemment has certain rights in this inven-tion.
~ield of the Invention This invention relates generally to the ~leld of Biology, and more particularly to the fields of Biomedicine, 8iochemistry and Molecular Biology.
Back~round and Summary of the Invention Mycoplasmas are a group of pathogenic microor~anisms of the Class Mollicutes characterized by having a small size and lacking a cell wall. These microol~anisms are among the FnlAllest-known organisms capable of a free living existence, and are important pathogens in man, -2- 1~39547 plants and animals. For example, atypical pneumonia and non-gonococcal urethritis are common mycoplasma infections in man. Mycoplasmas have also been associated with rheumatoid arthritis, spontaneous abortion, infer-tility and other genital tract diseases, and certain autoimmune disease states. Moreover, mycoplasmas are common contaminants in cell cultures. In biological research, mycoplasma contamination of tissue culture is a serious problem which demands constant monitoring.
Not surprisingly, these organisms are extremely fastidious and at present there are no cost-effective specific diagnostic procedures to determine the presence of mycoplasma infections. The most commonly-employed detection methods for mycoplasmas in clinical samples are serological and cultural. The serological methods are subject to false positives and the cultural methods are costly, time consuming and tedious.
Many of the biochemical techniques in current usage for detection of microbial contaminants in cell cultures do not specifically detect myco-plasmas but rather indicate the presence of any prokaryote or simple eukaryote such as yeast and fungi, and some may even detect viruses. Such a test is advantageous if one is interested only in the knowledge that a microbial agent is present, but if one is searching for a suspected eti~
logical agent of an animal or human disease it is obviously necess&ry to classify the agent as fully as possible.
Further, the above proced~l,es are hampered by special prob-lems. ~or example, there are apparently "non-cultivable" mycopl~smas which are not detected by conventional culture methods. In addition, in the case of immunofluorescence tests more than one antibody might be re -3- 1339~547 quired to identify the particular organism since more than nine different mycoplasma species are common tissue culture cont~min~nts. Also, DNA
stains are not necessarily mycoplasma-specific.
Therefore, a simple, sensitive, specific, cost-effective, snd rapid mycopl~sma detection system has been a desideratum in the fields of diagnostic medicine and biological research.
The use of nucleotide sequence homology snd nucleic acid hybridization kinetics has become a widely-employe~d technique for detect-ing various organisms in cells and cell cultures. However, prior to this invention reliable and specific DNA probes have not been Qvailable for mycoplasma detection.
The present invention proceeds by the use of specific myco-pl~sma ribosomal RNA gene fragments which are lsbeled or tsgged by a variety of techniques, such as rsdioisotope l~beling, biotin labeling, PEI-peroxidase conjugates, or nuorescent antibody tagging ELISA methods, for the specific and sensitive detection of mycoplasmas in clinical specimens, cells or cell cultures by DNA or RNA hybridization.
In one sspect of the invention, a DNA sequence from the 16S
RNA gene of mycoplasma is provided, which includes a nucleotide sequence selected from the group consisting of AACACGTATC, CGAATCAGCTATGTCG, GAGGTT AAC, ATCCGGATTTATT, TCTCAGTTCGGATTGA, AGGTGGTGCATGGTTG, TCCTGGCTCAGGAT, ATACATAGGT, AACTATGTGC, AA ~ l~ACAATG, snd TCTCGGGTCT, which code for mycoplasma ribosomal RNA (rRNA) where T represents thymine, G represcnt guanine, A represents adenine, C repre-4~ 1339a~7 sents cytosine and - indicates a nucleotide deletion within the sequence with respect to the comparable sequence in E. coli. These fragments differ significantly from the 16S RNA gene of E. ~? and thus form the basis for mycoplasma-specific probes which are constituted of labeled nucleotide sequences complementary to the above.
In another aspect of the invention, identified DNA sequences of a 16S RNA gene are provided which include nucleotide sequences selected from the group consisting of ACGGGTGAGT, TAATACCGCAT, TACGGGAGGCAGCAGT, GTGGGGAGCAAA, AGGATTAGATACCCT, CCGTAAACGAT, GAATTGACGGGG, CCCGCACAAG, GGTGGAGCATGT, TGTTGGGTTAAGTCCCGCAACGA, GGGATGACGT, ACGTGCTACAATG, CTAGTAATCG, TGTACACACCGCCCGTCA, AAGTCGTAACAAGGTA, and TGGATCACCTCCTT, which code for prokaryotic rRNA. These frag-ments represent regions within the 16S RNA gene that are identical for E.
coli and all mycoplasmas examined. Universal probes for all prokaryotes are constituted of labeled nucleotide sequences complementary to these fragments.
In general the invention comprises a method for determining the presence of a prokaryotic organism which contains a nucleic acid including a particular nucleotide sequence which is present in nucleic acids from prokaryotic organisms but absent in nucleic acids from eukaryotic organ-isms, which comprises contacting a medium which may contain a nucleic acid or nucleic acid fragment from s~id prokaryotic organism including said particular nucleotide ~quence with an oligonucleotide, including a nucleo-tide sequence complementary to said particular nucleotide ~equence, 1339~47 whereby said oligonucleotide hybridizes with any nucleic acid or nucleic acid fragment from said prokaryotic organism, including said particular nucleotide sequence which may be present in said medium, and detecting the presence of any nucleic acid or nucleic acid fragment hybridized with said oligonucleotide.
Other aspects of the invention c~nccll. the specific biological probes used for detecting mycoplasmss or prokaryotes in general in accord-ance with the above described proce~ and the identification and production of such probes.
The sole figure of the drawing shows a slot blot development illustrating the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention will now be described by detailing first the spe-cific steps involved in producing and using the biological probes of the present invention and then describing how particular nucleotide sequences useful in this invention are determined.
SYNTHESIS OF DEOXYOLIGONUCLEOTIDES
The 16 bp deoxyoligonucleotide, GCTTAGTCGATACAGC, which is complementary to one of the 16S mycoplasma RNA gene sequences listed above, was synthesized using the phosphotriester solid phase procedure described in M. H. C~ruthers et aL, Genetic Engineerin~, -6- 1339~47 Vol. 4, p. 1-17, Plenum Publishing Co. (1982).
Any of the other deoxyoligonucleotides previously mentioned or any other desired oligonucleotide can be similarly prepared. For example, instead of a DNA probe it may be desired to synthesize an RNA probe such as a recombinant SP6 vector transcript containing the sequence CGAAUCAGCUAUGUCG.
DNA-RNA or RNA-RNA hybridization to ribosomal RNA mole-cules amplifies the sensitivity of the detection several hundredfold above any DNA-DNA or RNA-DNA hybridization using probes against genomic DNA sequences, since the use of such probe which will detect multiple copies of ribosomal RNA per mycoplasma or eubacterial cell.
TESTING OF DEOXYOLIGONUCLEOTIDES
The above described deoxyoligonucleotide was 32P-labeled at the 5' end using the procedure in C. C. Richardson, Procedures in Nucleic Acid Research (Cantoni, G. L. and Davies, D. R., eds.), Vol. 2, pp. 815-828, Harper and Row, New York (1971). The resultin~ labeled deoxyoligonucleotide was then used as a myco-plasma-specific probe. Specificity for mycoplasma was demonstrated by means of slot blot hyL. i~ ation as described in M. Cunningham, Anal.
Biochem. 128:41~421 l1983~.
For this purpose a 1600 bp DNA fragment of Mycoplasma pneumoniae which hsd been cloned into pUC8 was used. The 1600 bp fragment contains the above described 16 base deoxyoligonucleotide. A genomic digest of E.
~, a representative prokasyotic eubacterium, was also produced by -7- 133~47 digestion with the enzyme Hindm. The digested E. coli DNA ~nd the 1600 bp M. pneumoniae DNA fragment were trsnsferred onto nitrocellulose filters according to the procedure in the J. J. Leary et sl., Proc. Natl.
Acad. Sci. U.S.A. 80:4045-4049 (19831 The nitroc~ filters containing the DNA fr~mPnt~ were baked for 2 hours at 80~C under reduced pressure and hybridized to the 32P-labeled deoxyoligonucleotide. Development of the resulting slot blots, shown in the drawing, revealed blots of increasing intensity for the M.
pneumoni~e DNA segment at 0.00034 ng, 0.0034 ng, 0.034 ng, 0.34 ng, and 3.4 ng (calculated for 16 bp) and no blots for the E. coli DNA segment at 0.00015 llg, 0.0015 ~Ig, 0.015 llg, 0.15 llg,and 1.5 llg indicating the sp~
cificity of the deoxyoligonucleotide probe for mycoplasma. The deoxy-oligonucleotide having the sequence GCTTAGTCGATACAGC thus is useful as a mycoplasma-specific probe which hybridizes with mycoplasmal DNA
but does not hybridize with DNA of other prokaryotic organisms. On the other hand, a deoxyoligonucleotide having the sequence TGCCCACTCA, for example, which is complementary to one of the prokaryotic coding gene sequences, is useful as a prokaryote-specific probe which hybridizes with prokaryotes but not with eukaryotes.
For the detection of mycoplasmas in infected cells the follow-ing procedure h~ ~een found ef~ective. The cells are trypsinized using 1-_ T75 tissue culture flasks with Trypsin EDTA (0.05% trypsin, 0.04~ EDTA
in PBS) for 2 minutes at 3~C. The trypsinized cells are resuspended in 1-2 ml of growth medium and spotted in a quantity of 50-100 Ill (1 x 105 -1 x 106 cells) onto a nitrocellulose filter wetted with 10 x SSC (1 x SSC:
~ -8- 1339~47 15 mM Na citrate, 150 mM NaCl, pH 7.4) using a Minifold I[ slot blot hybri-dization apparatus available from Schleicher and Schuell, Inc., Keene, N. H. The DNA samples applied to the slot blots are denatured with alkali (0.5 M NaOH, 1.5 M NaCl) for 5-lOminutes at room temperature and neu-tralized for 5-10 minutes at room temperature using 0.5 M Tris, pH 7.2 and 3.0 M NaCl. The filter is then washed with 2 x SSC for five minutes at room temperature and baked in a vacuum oven for 2 hours at 80~C. The filter is prehybridized for 2 hours at 65~C using a prehybridization buffer consisting of 0.5 mM EDTA, 5 mM Tris, pH 7.5, 5 x Denhardt, and 100 ~g/ml heat denatured her,in~ sperm DNA. Hybridization, using the probes of this invention in a concentration measured as 1-2 x 106 cpm of 32P-labeled deoxyoligonucleotide, specific sctivity >108 cpm/~l g in hybridi-zation buffer consisting of 10 mM Tris, pH 7.5,1 mM EDTA, 0.75 M NaCl, 1 x Denhardt, 0.5% SDS, 10% dextran sulfate and 100 llg/ml heat dena-tured herring sperm DNA, is carried out at 65~C for 16 hours. Following hybridization the filter is washed 2-4 hours at 65~C with 2 x SET, 0.2% SDS
(1 x SET:30 mM Tris, pH 8.0, 150 mM NaCl) and 1-2 hours at room tem-perature with 4 mM Tris base. The Blter is then dried and exposed on X-ray film using 1 or 2 Dupont Cronex intensifying scree1ls.
DETERMINATION OF PARTICULAR NVCLEOTIDE SEQUENCES
While the foregoing description of the present invention teaches how particular nucleotide seguences can be prepared and used, the broader scope of this invention may be re~li7ed by ~y~minirlg the technigues used for determining particular nucleotide sequences which are useful as myc~
1~39~54 7 g plasma-specific probes, probes specific for prokaryotes in general, probes specific for individual mycoplasma, ureaplasma, acholeplasma, and spir~
plasma species or probes specific for individual eubacterial species.
Such determination involves the following steps:
1. cloning the entire genome of ribosomal RNA of a particul~r species of mycoplasma into a bacteriophage or plasmid vector;
2. probing the resulting ribosomal RNA gene fragments with a non-mycoplasm~ prokaryotic ribosomal RNA operon;
3. characterizing the fragments which hybridize with said non-mycoplasma prokaryotic ribosomal RNA operon;
4. identifying mycoplasma~pecific fragments by differential hybridization as described in Gobel, U. and Stanbridge, E. J., Science, VoL
226, pp. 1211-12~3 (1984).
5. subcloning mycoplasma-specific fragments into a sequencing plasmid;
6. sequencing the resulting subcloned mycoplasma-specific fragments;
7. repeating steps 1-6 for other species of mycoplasma and for non-mycopl~smal prokaryotes; and - 8. comparing sequences obtained in steps 6 and 7; whereby a sequence common to all the species of mycoplasma but differing from the corresponding sequence in non-mycopl~rnsl prokaryotes is useful as a mycoplasma specific pro~e ~ 8 sequence comm~n to all the species of mycoplasm&, ~ ell ~s the non-mycoplasmal ptokaryotes, is useful as a probe specific for prokaryotes in general, and a sequence specific for either ~, 1339~47 --1 o a specific mycoplasms, acholeplasma, ureaplssma, or spiropl~sma species and sequences specific for any given eubacterial species, are useful 8S
probes specific for individual mycoplasma, acholeplasma, ureaplasma, spiroplasma, or eubacterial species, respectively.
Cloning of the ribosomal RNA genome of M. pneumoniae was accomplished by Hindm digestion of total M. pneumoniae DNA and ligation of the Hindm fragments to the Hindm digested vector pUC8. The resulting ribosomal RNA gene fragments were probed with E. coli ribosomal RNA
operon in the pKK 3535 plasmid according to the procedure in Gobel et al., Science, 226:1211-1213 (1984) to identify cloned fragments which contained ribosomal sequences. A
1600 bp fragment was chosen on the basis of hybridization to mycoplasma species and not to E. coli or mammalian DNA under stringent hybridization conditions. This 1600 bp fragment was removed from the pUC8 vector by means of HindI~ digestion and ligated to M13Mp8 DNA bacterial virus for sequencing using the Sanger dideoxy method described in Sanger et al., Proc. Natl. Acad. Sci. U.S.A., 74:5463-5467 (1977).
Comparison of the sequences of the mycoplasma species M.
pneumoniae, M. capricolum, snd Mycoplasma spe~ies PG50 with E. coli indicated that cert~in sequen~es were common to all these species of mycoplasrn~ b~t di~~erent from E. coli. These sequences could be synthe-sized and labeled and used as mycoplasma-specific probes. For example, GCTTAGTCGATACAGC constitued a mycoplasma-specific probe. Other sequences were common to these species of mycoplasma as well as E.
coli. These latter sequences could be synthesized, labeled, and used as probes specific for all prokaryotic species. Still other sequences were unique to a single mycoplasma species and could be synthesized, labeled, and used as mycoplasma species-specific probes.
The present invention thus provides a specific, sensitive, and rapid method for the detection of mycoplasmas in contaminated cell cul-tures or other biological environments. Alternatively, the present inven-tion can be used to provide a ribosomal DNA probe derived from a domain conserved in all prokaryotes. Such a probe would be ex-tremely useful in the rapid and sensitive diagnosis of a bacteremiH or septicemia in man or QnimQl~. The present invention may also be used to provide ribosomal DNA
probes that are specific for individual mycoplasma, acholeplasma, ures-plasma, spiroplasma, and eubacterial species, respectively. These probes will be of particular use for those organisms where little or no information exists on their genetic make-up.
Although the present invention has been described in detail by reference to certain specific examples of deoxyoligonucleotides and myco-plasma species, it should be apparent to one skilled in the art that various modifications are possible. It is intended that this invention include such modifications and that the invention be limited only in accordance with the claims appended hereto.
probes specific for individual mycoplasma, acholeplasma, ureaplasma, spiroplasma, or eubacterial species, respectively.
Cloning of the ribosomal RNA genome of M. pneumoniae was accomplished by Hindm digestion of total M. pneumoniae DNA and ligation of the Hindm fragments to the Hindm digested vector pUC8. The resulting ribosomal RNA gene fragments were probed with E. coli ribosomal RNA
operon in the pKK 3535 plasmid according to the procedure in Gobel et al., Science, 226:1211-1213 (1984) to identify cloned fragments which contained ribosomal sequences. A
1600 bp fragment was chosen on the basis of hybridization to mycoplasma species and not to E. coli or mammalian DNA under stringent hybridization conditions. This 1600 bp fragment was removed from the pUC8 vector by means of HindI~ digestion and ligated to M13Mp8 DNA bacterial virus for sequencing using the Sanger dideoxy method described in Sanger et al., Proc. Natl. Acad. Sci. U.S.A., 74:5463-5467 (1977).
Comparison of the sequences of the mycoplasma species M.
pneumoniae, M. capricolum, snd Mycoplasma spe~ies PG50 with E. coli indicated that cert~in sequen~es were common to all these species of mycoplasrn~ b~t di~~erent from E. coli. These sequences could be synthe-sized and labeled and used as mycoplasma-specific probes. For example, GCTTAGTCGATACAGC constitued a mycoplasma-specific probe. Other sequences were common to these species of mycoplasma as well as E.
coli. These latter sequences could be synthesized, labeled, and used as probes specific for all prokaryotic species. Still other sequences were unique to a single mycoplasma species and could be synthesized, labeled, and used as mycoplasma species-specific probes.
The present invention thus provides a specific, sensitive, and rapid method for the detection of mycoplasmas in contaminated cell cul-tures or other biological environments. Alternatively, the present inven-tion can be used to provide a ribosomal DNA probe derived from a domain conserved in all prokaryotes. Such a probe would be ex-tremely useful in the rapid and sensitive diagnosis of a bacteremiH or septicemia in man or QnimQl~. The present invention may also be used to provide ribosomal DNA
probes that are specific for individual mycoplasma, acholeplasma, ures-plasma, spiroplasma, and eubacterial species, respectively. These probes will be of particular use for those organisms where little or no information exists on their genetic make-up.
Although the present invention has been described in detail by reference to certain specific examples of deoxyoligonucleotides and myco-plasma species, it should be apparent to one skilled in the art that various modifications are possible. It is intended that this invention include such modifications and that the invention be limited only in accordance with the claims appended hereto.
Claims (5)
1. A method for determining the presence of a prokaryotic organism which contains a nucleic acid including a particular nucleotide sequence which is present in nucleic acids from prokaryotic organisms but absent in nucleic acids from eukaryotic organisms, which comprises:
contacting a medium which may contain a nucleic acid or nucleic acid fragment from said prokaryotic organism including said particular nucleotide sequence with an oligonucleotide including a nucleotide sequence complementary to said particular nucleotide sequence, whereby said oligonucleotide hybridizes with any nucleic acid or nucleic acid fragment from said prokaryotic organism including said particular nucleotide sequence which may be present in said medium; and detecting the presence of any nucleic acid or nucleic acid fragment hybridized with said oligonucleotide;
characterized in that said particular nucleotide sequence includes at least one of the following sequences or a sequence complementary to at least oneof the following sequences: TAGATATATG, AACACGTATC, CGAATCAGCTATGTCG, GAGGTT-AAC, ATCCGGATTTATT, TCTCAGTTCGGATTGA, AGGTGGTGCATGGTTG, TCCTGGCTCAGGAT, ATACATAGGT, AACTATGTGC, AATTTTTCACAATG, TCTCGGGTCT, ACGGGTGAGT, TAATACCGCAT, TACGGGAGGCAGCAGT, GTGGGGAGCAAA, AGGATTAGATACCCT, CCGTAAACGAT, GAATTGACGGGG, CCCGCACAAG, GGTGGAGCATGT, TGTTGGGTTAAGTCCCGCAACGA, GGGATGACGT,ACGTGCTACAATG, CTAGTAATCG, TGTACACACCGCCCGTCA, AAGTCGTAACAAGGTA and TGGATCACCTCCTT, wherein T represents thymine, G represents guanine, A
represents adenine, C represents cytosine and - indicates a nucleotide deletion within the sequence.
contacting a medium which may contain a nucleic acid or nucleic acid fragment from said prokaryotic organism including said particular nucleotide sequence with an oligonucleotide including a nucleotide sequence complementary to said particular nucleotide sequence, whereby said oligonucleotide hybridizes with any nucleic acid or nucleic acid fragment from said prokaryotic organism including said particular nucleotide sequence which may be present in said medium; and detecting the presence of any nucleic acid or nucleic acid fragment hybridized with said oligonucleotide;
characterized in that said particular nucleotide sequence includes at least one of the following sequences or a sequence complementary to at least oneof the following sequences: TAGATATATG, AACACGTATC, CGAATCAGCTATGTCG, GAGGTT-AAC, ATCCGGATTTATT, TCTCAGTTCGGATTGA, AGGTGGTGCATGGTTG, TCCTGGCTCAGGAT, ATACATAGGT, AACTATGTGC, AATTTTTCACAATG, TCTCGGGTCT, ACGGGTGAGT, TAATACCGCAT, TACGGGAGGCAGCAGT, GTGGGGAGCAAA, AGGATTAGATACCCT, CCGTAAACGAT, GAATTGACGGGG, CCCGCACAAG, GGTGGAGCATGT, TGTTGGGTTAAGTCCCGCAACGA, GGGATGACGT,ACGTGCTACAATG, CTAGTAATCG, TGTACACACCGCCCGTCA, AAGTCGTAACAAGGTA and TGGATCACCTCCTT, wherein T represents thymine, G represents guanine, A
represents adenine, C represents cytosine and - indicates a nucleotide deletion within the sequence.
2. A method according to Claim 1, characterized in that said particular nucleotide sequence includes at least one of the following mycoplasma-specific sequences or a sequence complementary to at least one of the following sequences: TAGATATATG, AACACGTATC, CGAATCAGCTATGTCG, GAGGTT-AAC, ATCCGGATTTATT, TCTCAGTTCGGATTGA, AGGTGGTGCATGGTTG, TCCTGGCTCAGGAT, ATACATAGGT, AACTATGTGC, AATTTTTCACAATG, and TCTCGGGTCT.
3. A method according to Claim 1, characterized in that said particular nucleotide sequence includes at least one of the following prokaryotic sequencesor a sequence complementary to at least one of the following sequences:
ACGGGTGAGT, TAATACCGCAT, TACGGGAGGCAGCAGT, GTGGGGAGCAAA, AGGATTAGATACCCT, CCGTAAACGAT, GAATTGACGGGG, CCCGCACAAG, GGTGGAGCATGT, TGTTGGGTTAAGTCCCGCAACGA, GGGATGACGT,ACGTGCTACAATG, CTAGTAATCG, TGTACACACCGCCCGTCA, AAGTCGTAACAAGGTA and TGGATCACCTCCTT.
ACGGGTGAGT, TAATACCGCAT, TACGGGAGGCAGCAGT, GTGGGGAGCAAA, AGGATTAGATACCCT, CCGTAAACGAT, GAATTGACGGGG, CCCGCACAAG, GGTGGAGCATGT, TGTTGGGTTAAGTCCCGCAACGA, GGGATGACGT,ACGTGCTACAATG, CTAGTAATCG, TGTACACACCGCCCGTCA, AAGTCGTAACAAGGTA and TGGATCACCTCCTT.
4. A mycoplasma-specific probe, characterized in that it hybridizes with a particular nucleotide sequence including at least one of the following mycoplasma-specific sequences or a sequence complementary to at least one of the following sequences: TAGATATATG, AACACGTATC, CGAATCAGCTATGTCG, GAGGTT-AAC, ATCCGGATTTATT, TCTCAGTTCGGATTGA, AGGTGGTGCATGGTTG, TCCTGGCTCAGGAT, ATACATAGGT, AACTATGTGC, AATTTTTCACAATG andTCTCGGGTCT.
5. A prokaryotic-specific probe, characterized in that it hybridizes with a particular nucleotide sequence including at least one of the following prokaryotic sequences or a sequence complementary to at least one of the following sequences: ACGGGTGAGT, TAATACCGCAT, TACGGGAGGCAGCAGT, GTGGGGAGCAAA, AGGATTAGATACCCT, CCGTAAACGAT, GAATTGACGGGG, CCCGCACAAG, GGTGGAGCATGT, TGTTGGGTTAAGTCCCGCAACGA, GGGATGACGT,ACGTGCTACAATG, CTAGTAATCG, TGTACACACCGCCCGTCA, AAGTCGTAACAAGGTAand TGGATCACCTCCTT.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000512288A CA1339547C (en) | 1986-06-24 | 1986-06-24 | Detection of mycoplasma by dna hybridization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000512288A CA1339547C (en) | 1986-06-24 | 1986-06-24 | Detection of mycoplasma by dna hybridization |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1339547C true CA1339547C (en) | 1997-11-18 |
Family
ID=4133415
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000512288A Expired - Lifetime CA1339547C (en) | 1986-06-24 | 1986-06-24 | Detection of mycoplasma by dna hybridization |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1339547C (en) |
-
1986
- 1986-06-24 CA CA000512288A patent/CA1339547C/en not_active Expired - Lifetime
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