Method and kit for identifying antibiotic-resistant microorganisms

• US 20040229268A1
• Filed: 05/11/2004
• Published: 11/18/2004
• Est. Priority Date: 05/13/2003
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Hogan, James J., Kaplan, Shannon K.
• Granted Patent: US 7,786,289 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

The invention provides a rapid sample-processing method for preparing hybridization reaction mixtures substantially depleted of RNA, and a method of identifying the methicillin-resistance status and vancomycin-resistance status of an organism.

Claims

1. A device for detecting nucleic acids encoding resistance to an antibiotic, comprising:

a solid support and a plurality of detectably labeled hybridization probes distributed among a plurality of loci thereon, said plurality of loci comprising,

a first locus that comprises one or more probes that collectively hybridize to ribosomal nucleic acids from a plurality of species of bacteria in the genus Staphylococcus, comprising Staphylococcus aureus and Staphylococcus epidermidis, but do not hybridize to ribosomal nucleic acids of bacteria in the genus Enterococcus,

a second locus that comprises

(a) one or more probes that collectively hybridize to ribosomal nucleic acids from a plurality of bacteria in the genus Enterococcus, comprising Enterococcus faecalis and Enterococcus faecium, but not Staphylococcus aureus or any other bacteria in the genus Staphylococcus, and

(b) at least one probe that hybridizes to ribosomal nucleic acids from Staphylococcus aureus but not from other species in the genus Staphylococcus or bacteria in the genus Enterococcus,

a third locus that comprises at least one probe that hybridizes to mecA nucleic acids, and

a fourth locus that comprises one or more probes that collectively hybridize to VanA nucleic acids and to VanB nucleic acids.

2. The device of claim 1, wherein each of said plurality of detectably labeled hybridization probes is a detectably labeled soluble hybridization probe.

3. The device of claim 1, wherein each of said detectably labeled hybridization probes is labeled with a homogeneously detectable label.

4. The device of claim 3, wherein said homogeneously detectable label is a chemiluminescent label.

5. The device of claim 1, further comprising a fifth locus, said fifth locus comprising one or more probes that collectively hybridize to ribosomal nucleic acids from a plurality of species of Gram-positive bacteria comprising the high(G+C) subset thereof, a plurality of species of bacteria in the family Enterobacteriaceae, a plurality of species of bacteria in the genus Enterococcus, and a plurality of species of bacteria in the genus Staphylococcus.

6. The device of claim 1, wherein said one or more probes that collectively hybridize to VanA nucleic acids and to VanB nucleic acids comprise at least one probe that hybridizes to VanA nucleic acids and at least one probe that hybridizes to VanB nucleic acids.

7. The device of claim 1, wherein said one or more probes that collectively hybridize to VanA nucleic acids and to VanB nucleic acids comprise a single probe that hybridizes both to VanA nucleic acids and VanB nucleic acids.

8. A device for detecting nucleic acids encoding resistance to an antibiotic, comprising:

a solid support and a plurality of detectably labeled hybridization probes distributed among a plurality of loci thereon, said plurality of loci comprising,

a first locus that comprises one or more probes that collectively hybridize to ribosomal nucleic acids from a plurality of species of bacteria in the genus Staphylococcus, comprising Staphylococcus aureus and Staphylococcus epidermidis,

a second locus that comprises a probe that hybridizes to ribosomal nucleic acids from Staphylococcus aureus but not other species in the genus Staphylococcus,

a third locus that comprises at least one probe that hybridizes to mecA nucleic acids, and

a fourth locus that comprises one or more probes that collectively hybridize to ribosomal nucleic acids from a plurality of species of Gram-positive bacteria comprising the high(G+C) subset thereof, a plurality of species of bacteria in the family Enterobacteriaceae, a plurality of species of bacteria in the genus Enterococcus, and a plurality of species of bacteria in the genus Staphylococcus.

9. The device of claim 8, wherein said one or more probes of said fourth locus comprise a pan-bacterial probe selected from the group consisting of the base sequence of SEQ ID NO:

16 and the base sequence of SEQ ID NO;

19.

10. The device of claim 9, wherein said at least one probe of said third locus has a base sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3 and SEQ ID NO;

5.

11. A device for detecting nucleic acids encoding resistance to an antibiotic, comprising:

a solid support and a plurality of detectably labeled hybridization probes distributed among a plurality of loci thereon, said plurality of loci comprising,

a first locus that comprises one or more probes that collectively hybridize to ribosomal nucleic acids from a plurality of bacteria in the genus Enterococcus, comprising Enterococcus faecalis and Enterococcus faecium,

a second locus that comprises one or more probes that collectively hybridize to VanA nucleic acids and to VanB nucleic acids, and

a third locus that comprises one or more probes that collectively hybridize to ribosomal nucleic acids from a plurality of species of Gram-positive bacteria comprising the high(G+C) subset thereof, a plurality of species of bacteria in the family Enterobacteriaceae, a plurality of species of bacteria in the genus Enterococcus, and a plurality of species of bacteria in the genus Staphylococcus.

12. The device of claim 11, wherein said one or more probes of said third locus comprise a pan-bacterial probe selected from the group consisting of SEQ ID NO:

16 and SEQ ID NO;

19.

13. The device of claim 12, wherein said one or more probes of said second locus have base sequences selected from the group consisting of SEQ ID NO:

27, SEQ ID NO;

28 and SEQ ID NO;

29.

14. The device of claim 12, wherein said one or more probes of said second locus have base sequences selected from the group consisting of SEQ ID NO:

30, SEQ ID NO;

40 and SEQ ID NO;

41.

15. A method of detecting the presence of a target DNA sequence in a sample containing cells, comprising the steps of:

lysing cells contained in the sample to yield a lysate;

treating the lysate with a basic composition to yield a basic mixture;

introducing one or more reagents to the basic mixture to yield a reaction mixture at a pH in the range of 4.5-5.5;

hybridizing the reaction mixture under high stringency hybridization conditions with an oligonucleotide probe to form a hybrid between the oligonucleotide probe and the target DNA; and

detecting said hybrid as an indicator of the presence of said target DNA sequence.

16. The method of claim 15, wherein said oligonucleotide probe is complementary either to a mecA gene sequence, a VanA gene sequence or a VanB gene sequence.

17. The method of claim 15, wherein said treating step comprises heating said basic mixture at a temperature up to 100°

C. for a time sufficient to substantially hydrolyze RNA that may be present in said lysate.

18. A probe mix for hybridizing mecA nucleic acids, comprising:

a first detectably labeled hybridization probe comprising the base sequence of SEQ ID NO;

2

a second detectably labeled hybridization probe comprising the base sequence of SEQ ID NO;

3

a third detectably labeled hybridization probe comprising the base sequence of SEQ ID NO;

5.

19. The probe mix of claim 18, wherein said first detectably labeled hybridization probe comprises the length and base sequence of SEQ ID NO:

2 or the complement thereof, and optionally a non-complementary sequence that does not hybridize to the nucleic acids of the mecA gene;

wherein said second detectably labeled hybridization probe comprises the length and base sequence of SEQ ID NO;

3 or the complement thereof, and optionally a non-complementary sequence that does not hybridize to the nucleic acids of the mecA gene;

wherein said third detectably labeled hybridization probe comprises the length and base sequence of SEQ ID NO;

5 or the complement thereof, and optionally a non-complementary sequence that does not hybridize to the nucleic acids of the mecA gene.

20. A probe mix for detecting VanA nucleic acids, comprising:

a first detectably labeled hybridization probe comprising the base sequence of SEQ ID NO;

27 or the complement thereof;

a second detectably labeled hybridization probe comprising the base sequence of SEQ ID NO;

28 or the complement thereof; and

a third detectably labeled hybridization probe comprising the base sequence of SEQ ID NO;

29 or the complement thereof.

21. The probe mix of claim 20, wherein said first detectably labeled hybridization probe comprises the length and base sequence of SEQ ID NO:

27 or the complement thereof, and optionally a non-complementary sequence that does not hybridize to the nucleic acids of the VanA gene;

wherein said second detectably labeled hybridization probe comprises the length and base sequence of SEQ ID NO;

28 or the complement thereof, and optionally a non-complementary sequence that does not hybridize to the nucleic acids of the VanA gene;

wherein said third detectably labeled hybridization probe comprises the length and base sequence of SEQ ID NO;

29 or the complement thereof, and optionally a non-complementary sequence that does not hybridize to the nucleic acids of the VanA gene.

22. A probe mix for detecting VanB nucleic acids, comprising:

a first detectably labeled hybridization probe comprising the base sequence of SEQ ID NO;

30 or the complement thereof;

a second detectably labeled hybridization probe comprising the base sequence of SEQ ID NO;

40 or the complement thereof; and

a third detectably labeled hybridization probe comprising the base sequence of SEQ ID NO;

41 or the complement thereof.

23. The probe mix of claim 22, wherein said first detectably labeled hybridization probe comprises the length and base sequence of SEQ ID NO:

30 or the complement thereof, and optionally a non-complementary sequence that does not hybridize to the nucleic acids of the VanB gene;

wherein said second detectably labeled hybridization probe comprises the length and base sequence of SEQ ID NO;

40 or the complement thereof, and optionally a non-complementary sequence that does not hybridize to the nucleic acids of the VanB gene;

wherein said third detectably labeled hybridization probe comprises the length and base sequence of SEQ ID NO;

41 or the complement thereof, and optionally a non-complementary sequence that does not hybridize to the nucleic acids of the VanB gene.