Methods for selectively detecting microorganisms associated with vaginal infections in complex biological samples

• US 5,700,636 A
• Filed: 10/08/1993
• Issued: 12/23/1997
• Est. Priority Date: 10/19/1990
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Stamm, Michael R., Cangelosi, Gerard A., Britschgi, Theresa B., Adams, Trevor H., Sheiness, Diana K., Dix, Connie K.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/133,598
• Time in Patent Office: 1,537 Days
• Field of Search: 435/6, 435/91.2, 536/24.32, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form

Abstract

The present invention relates, in general, to methods for selectively detecting a prokaryotic microorganism and a eukaryotic microorganism in a single complex biological sample wherein the cells of such microorganisms are lysed by combining the sample with a lysis solution and contacting the nucleic acid released from the microorganisms with selective nucleic acid probes through hybridization techniques. Further provided are methods which permit the detection of a Gram-positive bacterium with at least one other microorganism selected from the group consisting of yeast, protozoa, mycoplasma and Gram-negative bacteria in a single complex biological sample. In other aspects of the invention, the methods disclosed permit the detection of microorganisms associated with vaginal disorders.

Claims

1. A method for selectively detecting a prokaryotic microorganism and a eukaryotic microorganism in a single sample, the method comprising:

(a) lysing the cells of the prokaryotic microorganism and the eukaryotic microorganism by combining the sample with a lysis solution, thereby releasing nucleic acid from the prokaryotic and the eukaryotic microorganisms;

(b) contacting the nucleic acid released from the microorganisms, under hybridizing conditions, with a first oligonucleotide capture probe that selectively hybridizes to the nucleic acid of the prokaryotic microorganism, wherein said first oligonucleotide capture probe has a sequence selected from the group consisting of SEQ ID NOS;

1-7, 17-28, 30, 33-35, 42-47, 51 and 52 and a second oligonucleotide capture probe that selectively hybridizes to the nucleic acid of the eukaryotic microorganism, wherein said second oligonucleotide capture probe has a sequence selected from the group consisting of SEQ ID NOS;

13 and 48-50 to form a prokaryotic microorganism-capture probe hybridization complex and a eukaryotic microorganism-capture probe hybridization complex, respectively; and

(c) detecting the hybridization complexes as an indication of the presence of the prokaryotic microorganism and the eukaryotic microorganism in the sample.

2. The method in accordance with claim 1 wherein the prokaryotic microorganism is Gardnerella vaginalis.

3. The method in accordance with claim 1 wherein the eukaryotic microorganism is selected from the group consisting of Trichomonas vaginalis and Candida albicans.

4. The method in accordance with claim 1 wherein the oligonucleotide probe specific for the prokaryotic microorganism is immobilized on a first capture bead and the oligonucleotide probe specific for the eukaryotic microorganism is immobilized on a second capture bead, both beads being immobilized on a dipstick comprising a nonporous solid support.

5. The method in accordance with claim 1 wherein the prokaryotic microorganism-capture probe hybridization complex is detected by a first signal oligonucleotide which is not complementary to the capture probe and which hybridizes to the nucleic acid of the prokaryotic microorganism, and the eukaryotic microorganism-capture probe hybridization complex is detected by a second signal oligonucleotide which is not complementary to the capture probe and which hybridizes to the nucleic acid of the eukaryotic microorganism.

6. A method for selectively detecting at least one Group I microorganism selected from the group consisting of gram positive bacteria and fungi, and at least one group II microorganism selected from the group consisting of protozoa, mycoplasmas and gram negative bacteria in a single sample, the method comprising:

(a) lysing the cells of the Group I microorganism and the Group II microorganism by combining the sample with a lysis solution, thereby releasing nucleic acid from the microorganisms;

(b) contacting the nucleic acid released from the microorganisms, under hybridizing conditions, with a first oligonucleotide capture probe that selectively hybridizes to the nucleic acid of the Group I microorganism, wherein said first oligonucleotide capture probe has a sequence selected from the group consisting of SEQ ID NOS;

13, 18-26 and 42-47 and a second oligonucleotide capture probe that selectively hybridizes to the nucleic acid of the Group II microorganism, wherein said second oligonucleotide capture probe has a sequence selected from the group consisting of SEQ ID NOS;

1-7, 27, 28, 30, 32-35 and 48-52 to form a Group I microorganism-capture probe hybridization complex, and a Group II microorganism-capture probe hybridization complex respectively; and

(c) detecting the hybridization complexes as an indication of the presence of the Group I microorganism and the Group II microorganism in the sample.

7. The method in accordance with claim 6 wherein the Group I microorganism is Group B Streptococci.

8. The method in accordance with claim 6 wherein the Group I microorganism is Candida albicans.

9. The method in accordance with claim 6 wherein the Group II microorganism is Gardnerella vaginalis.

10. The method in accordance with claim 6 wherein the Group II microorganism is Trichomonas vaginalis.

11. The method in accordance with claim 6 wherein the oligonucleotide probe specific for the Group I microorganism is immobilized on a first capture bead and the oligonucleotide probe specific for the Group II microorganism is immobilized on a second capture bead, the capture beads being immobilized on a dipstick comprising a nonporous solid support.

12. The method in accordance with claim 6 wherein the Group I microorganism-capture probe hybridization complex is detected by a first signal oligonucleotide which is not complementary to the capture probe and which hybridizes to the nucleic acid of the Group I microorganism, and the Group II microorganism-capture probe hybridization complex is detected by a second signal oligonucleotide which is not complementary to the capture probe and which hybridizes to the nucleic acid of the Group II microorganism.

13. A method for selectively detecting Gardenerella vaginalis and at least one Group B microorganism selected from the group consisting of Group B Streptococci, Candida albicans and Trichomonas vaginalis in a single sample, the method comprising:

(a) lysing the cells of Gardenerella vaginalis and the Group B microorganism by combining the sample with a lysis solution, thereby releasing nucleic acid from the microorganisms;

(b) contacting the nucleic acid released from the microorganisms, under hybridizing conditions, with a first oligonucleotide capture probe that selectively hybridizes to the nucleic acid of Gardenerella vaginalis, wherein said first oligonucleotide capture probe has a sequence selected from the group consisting of SEQ. ID NOS;

18-26 and a second oligonucleotide capture probe that selectively hybridizes to the nucleic acid of the Group B microorganism, wherein said second oligonucleotide capture probe has a sequence selected from the group consisting of SEQ. ID NOS;

13 and 42-50 to form a Gardenerella vaginalis microorganism-capture probe hybridization complex and a Group B microorganism-capture probe hybridization complex, respectively; and

(c) detecting the hybridization complexes as an indication of the presence of the Gardenerella vaginalis microorganism and the Group B microorganism in the sample.