# US 5,700,636 A · Methods for selectively detecting microorganisms associated with vaginal infections in complex biological samples

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- Number: US 5,700,636 A
- Document ID: US5700636A
- Status: Granted
- Kind: A
- Application number: 08/133,598
- Filed: 1993-10-08
- Granted: 1997-12-23
- Earliest claimed: 1990-10-19
- Assignees: Becton, Dickinson and Company
- Inventors: Diana K. Sheiness; Trevor H. Adams; Michael R. Stamm; Gerard A. Cangelosi; Theresa B. Britschgi; Connie K. Dix
- Web page: https://patentgrep.com/patent/US5700636A

## CPC

- 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
- C12Q 1/6813: Hybridisation assays
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/689: for bacteria
- C12Q 1/6895: for plants, fungi or algae
- G01N 33/54313: {the carrier being characterised by its particulate form}
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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.

### Claim 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.

### Claim 7 (depends on 6)

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

### Claim 8 (depends on 6)

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

### Claim 9 (depends on 6)

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

### Claim 10 (depends on 6)

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

### Claim 11 (depends on 6)

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.

### Claim 12 (depends on 6)

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.

### Claim 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.