Nucleic acid probes for the detection of mycoplasma pneumoniae and mycoplasma genitalium

• US 5,552,279 A
• Filed: 07/29/1994
• Issued: 09/03/1996
• Est. Priority Date: 03/22/1991
• Status: Expired due to Term

• Current Assignee: Amoco Corporation (BP PLC)
• Original Assignee: Amoco Corporation (BP PLC)
• Inventors: Weisburg, William G., Pelletier, Dale A.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Schreiber, David
• Application Number:
  US08/283,067
• Time in Patent Office: 767 Days
• Field of Search: 435/6, 536/24.32
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

Nucleic acid probes are described for detecting the principle etiological agent of primary atypical pneumonia, Mycoplasma pneumoniae, or, optionally, Mycoplasma pneumoniae and Mycoplasma genitalium. Said probes are complementary to ribonucleic acid sequences found in these mycoplasmas and absent from other mycoplasma, other bacterial, animal, or plant genomes. As such, these probes can detect the rRNA, rDNA, or polymerase chain reaction amplification products from these mycoplasma species. This set of probes, plus the described amplification primers, circumscribe a method for detecting the etiological agents of atypical pneumonia, and for making a clinical diagnosis of this disease. This set of probes also circumscribes a method for identification of these infectious agents in culture media enrichments inoculated from clinical samples.

Claims

1. An isolated nucleic acid consisting of a nucleotide sequence fully complementary or identical to any one of probes 2295 (SEQ ID NO:

1), 2289 (SEQ ID NO;

2), 2296 (SEQ ID NO;

3), 2298 (SEQ ID NO;

4), 2299 (SEQ ID NO;

5), 2300 (SEQ ID NO;

6), and 2294 (SEQ ID NO;

7), and wherein said nucleic acid hybridizes to the 23S ribosomal RNA (rRNA) or ribosomal DNA (rDNA) of Mycoplasma pneumoniae under normal hybridization conditions and does not hybridize to rRNA or rDNA of non-Mycoplasma pneumoniae bacteria and humans under identical hybridization conditions.

2. A nucleic acid of claim 1, wherein said nucleic acid is fully complementary or identical to any one of probes 2295 (SEQ ID NO:

1), 2296 (SEQ ID NO;

3), 2298 (SEQ ID NO;

4), and 2299 (SEQ ID NO;

5).

4. A set of nucleic acids comprising at least two nucleic acids of claim 1.

5. A set of nucleic acids of claim 4 comprising probes(SEQ ID NO:

5) and 2300 (SEQ ID NO;

6).

6. A method for detecting the presence of Mycoplasma pneumoniae in a sample comprising:

a) contacting said sample with at least one nucleic acid of claim 1;

b) imposing hybridization conditions on the sample and said nucleic acid to allow said nucleic acid to hybridize to the rRNA or rDNA of Mycoplasma pneumoniae, if present in the sample, to form nucleic acid complexes, under conditions which do not allow said nucleic acid to form stable hybridized nucleic acid complexes with non-Mycoplasma bacteria; and

c) detecting said nucleic acid complexes as an indication of the presence of Mycoplasma pneumoniae in the sample.

15. A probe consisting of a nucleic acid of claim 1 carrying one or more of an end cap, a detection moiety, an amplifiable Q-beta midivariant and a homopolymer nucleotide sequence.

19. A probe consisting of a nucleic acid of claim 1 carrying an amplifiable Q-beta midivariant.

3. An isolated nucleic acid consisting of a nucleotide sequence fully complementary or identical to probe 2297 (SEQ ID NO:

10), and wherein said nucleic acid hybridizes to the 23S ribosomal RNA (rRNA) or ribosomal DNA (rDNA) of Mycoplasma pneumoniae and Mycoplasma genitalium under normal hybridization conditions and does not hybridize to rRNA or rDNA of non-Mycoplasma pneumoniae and non-Mycoplasma genitalium bacteria and humans under identical hybridization conditions.

16. A probe consisting of a nucleic acid of claim 3 carrying one or more of an end cap, a detection moiety, an amplifiable Q-beta midivariant and a homopolymer nucleotide sequence.

20. A probe consisting of a nucleic acid of claim 3 carrying an amplifiable Q-beta midivariant.

7. An isolated nucleic acid consisting of a nucleotide sequence fully complementary or identical to any one of probes 2162 (SEQ ID NO:

13), 2166 (SEQ ID NO;

16), and 2167 (SEQ ID NO;

8), which nucleic acid hybridizes to the 16S rRNA or rDNA of Mycoplasma pneumoniae or Mycoplasma genitalium under normal hybridization conditions and does not hybridize to the ribosomal RNA (rRNA) or ribosomal DNA (rDNA) of non-Mycoplasma pneumoniae and non-Mycoplasma genitalium bacteria and humans under identical hybridization conditions.

8. A nucleic acid of claim 7, wherein said nucleic acid is fully complementary or identical to any one of probes 2166 (SEQ ID NO:

16), and 2167 (SEQ ID NO;

8).

10. A set of nucleic acids comprising at least two nucleic acids, each nucleic acid having a different nucleotide sequence composition, and wherein a first nucleic acid is a nucleic acid of claim 7, and a second nucleic acid consists of a nucleotide sequence fully complementary or identical to any one of probes 2196 (SEQ ID NO:

9), 2192 (SEQ ID NO;

11), 2219 (SEQ ID NO;

12), 2202 (SEQ ID NO;

14), 2201 (SEQ ID NO;

15), 2224 (SEQ ID NO;

17), 2195 (SEQ ID NO;

18), 2225 (SEQ ID NO;

19), and 2230 (SEQ ID NO;

20), and wherein said second nucleic acid hybridizes to the 16S rRNA or rDNA of Mycoplasma pneumoniae or Mycoplasma genitalium under normal hybridization conditions and does not hybridize to the rRNA or rDNA of non-Mycoplasma pneumoniae and non-Mycoplasma genitalium bacteria and humans under identical hybridization conditions.

11. A set of nucleic acids of claim 10 selected from the group of sets:

2196 (SEQ ID NO;

9) and 2167 (SEQ ID NO;

8),

2162 (SEQ ID NO;

13) and 2202 (SEQ ID NO;

14),

2224 (SEQ ID NO;

17) and 2166 (SEQ ID NO;

16),

2162 (SEQ ID NO;

13) and 2230 (SEQ ID NO;

20), and

2167 (SEQ ID NO;

8) and 2219 (SEQ ID NO;

12).

12. A method for detecting the presence of Mycoplasma pneumoniae in a sample comprising:

a) contacting said sample with at least one nucleic acid of claim 7;

b) imposing hybridization conditions on the sample and said nucleic acid to allow said nucleic acid to hybridize to the rRNA or rDNA of Mycoplasma pneumoniae, if present in the sample, to form nucleic acid complexes, under conditions which do not allow said nucleic acid to form stable hybridized nucleic acid complexes with non-Mycoplasma bacteria; and

c) detecting said nucleic acid complexes as an indication of the presence of Mycoplasma pneumoniae in the sample.

13. A method for detecting the presence of either one or both of Mycoplasma pneumoniae and Mycoplasma genitalium in a sample comprising:

a) contacting said sample with a set of at least two nucleic acids of claim 10;

b) imposing hybridization conditions on the sample and said nucleic acids to allow said nucleic acids to hybridize stably to the rRNA or rDNA of either one or both of Mycoplasma pneumoniae and Mycoplasma genitalium, if present in the sample, to form nucleic acid complexes, under conditions which do not allow said probe to form stable hybridized nucleic acid complexes with non-Mycoplasma bacteria; and

c) detecting said nucleic acid complexes as an indication of the presence of either one or both of Mycoplasma pneumoniae and Mycoplasma genitalium in the sample.

14. A method of claim 13, wherein said set of nucleic acids is selected from the group of sets(SEQ ID NO:

9) and 2167 (SEQ ID NO;

8),

2162 (SEQ ID NO;

13) and 2202 (SEQ ID NO;

14),

2224 (SEQ ID NO;

17) and 2166 (SEQ ID NO;

16),

2162 (SEQ ID NO;

13) and 2230 (SEQ ID NO;

20), or

2167 (SEQ ID NO;

8) and 2219 (SEQ ID NO;

12).

17. A probe consisting of a nucleic acid of claim 7 carrying one or more of an end cap, a detection moiety, an amplifiable Q-beta midivariant and a homopolymer nucleotide sequence.

21. A probe consisting of a nucleic acid of claim 7 carrying an amplifiable Q-beta midivariant.

9. A nucleic acid that is fully complementary or identical to any one of probes 2219 (SEQ ID NO:

12), 2202 (SEQ ID NO;

14), 2225 (SEQ ID NO;

19), and 2230 (SEQ ID NO;

20).

18. A probe consisting of a nucleic acid of claim 9 carrying one or more of an end cap, a detection moiety, an amplifiable Q-beta midivariant and a homopolymer nucleotide sequence.

22. A probe consisting of a nucleic acid of claim 9 carrying an amplifiable Q-beta midivariant.