Method and kit for identifying antibiotic-resistant microorganisms

• US 9,109,261 B2
• Filed: 05/05/2010
• Issued: 08/18/2015
• 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.
• Primary Examiner(s): HANEY, AMANDA MARIE
• Application Number:
  US12/774,229
• Publication Number:
  US 20100216155A1
• Time in Patent Office: 1,931 Days
• Field of Search: None
• US Class Current: 1/1
• 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 solution-phase hybridization probes distributed among a plurality of loci thereon, said plurality of loci comprising,

(a) a first locus that comprises one or more solution-phase hybridization 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,

(b) a second locus that comprises a probe mix containing

(i) one or more solution-phase hybridization 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

(ii) at least one solution-phase hybridization 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,

(c) a third locus that comprises a probe mix containing a first mecA solution-phase hybridization probe that consists of the nucleotide sequence of SEQ ID NO:

2 or the complement thereof, a second mecA solution-phase hybridization probe that consists of the nucleotide sequence of SEQ ID NO;

3 or the complement thereof, and a third mecA solution-phase hybridization probe that consists of the nucleotide sequence of SEQ ID NO;

5 or the complement thereof, and

(d) a fourth locus that comprises a solution-phase hybridization probe that hybridizes both to VanA nucleic acids and VanB nucleic acids, wherein said solution-phase hybridization probe consists of the nucleotide sequence of SEQ ID NO;

33 or the complement thereof;

wherein each of said plurality of loci is configured as a matrix that provides an identification of one or more unknown nucleic acids from a sample, and wherein each of said detectably labeled solution-phase hybridization probes comprises a polynucleotide sequence which is covalently attached to a non-nucleotide detectable moiety that emits a detectable signal.

2. The device of claim 1, wherein said non-nucleotide detectable moiety is a homogeneously detectable label.

3. The device of claim 2, wherein said homogeneously detectable label is a chemiluminescent label.

4. The device of claim 1, further comprising a fifth locus, said fifth locus comprising one or more solution-phase hybridization 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.

5. The device of claim 1, wherein the first mecA solution-phase hybridization probe of said probe mix consists of the nucleotide sequence of SEQ ID NO:

2, wherein the second mecA solution-phase hybridization probe of said probe mix consists of the nucleotide sequence of SEQ ID NO;

3, and wherein the third mecA solution-phase hybridization probe of said probe mix consists of the nucleotide sequence of SEQ ID NO;

5.

6. The device of claim 1, wherein each of said plurality of detectably labeled solution-phase hybridization probes comprises the non-nucleotide detectable moiety prior to contact with a biological sample to be tested for the presence of microorganisms or nucleic acids thereof.

7. The device of claim 1, wherein said plurality of loci are wells of a multi-well plate.

8. A kit for differentiating between bacteria, wherein said kit comprises the device of claim 1.

9. The kit of claim 8, wherein said kit comprises one or more of, a specimen lysis reagent;

a probe hybridization reagent;

a selection reagent;

an alkaline hydrolysis reagent;

or an internal control nucleic acid.

10. A device for detecting nucleic acids encoding resistance to an antibiotic, comprising a solid support and a plurality of detectably labeled solution-phase hybridization probes distributed among a plurality of loci thereon, said plurality of loci comprising,

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

(b) a second locus that comprises a solution-phase hybridization probe that hybridizes to both VanA nucleic acids and to VanB nucleic acids, wherein said solution-phase hybridization probe consists of the nucleotide sequence of SEQ ID NO:

33 or the complement thereof, and

(c) a third locus that comprises one or more solution-phase hybridization 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;

wherein each of said plurality of loci is arranged as a matrix for identification of one or more soluble unknown nucleic acids from a sample, and wherein each of said detectably labeled solution-phase hybridization probes comprises a polynucleotide sequence which is covalently attached to a non-nucleotide detectable moiety that emits a detectable signal.

11. The device of claim 10, wherein said one or more solution-phase hybridization probes of said third locus comprise a pan-bacterial probe of SEQ ID NO:

16.

12. The device of claim 10, wherein each of said plurality of detectably labeled solution-phase hybridization probes comprises the non-nucleotide detectable moiety prior to contact with a biological sample to be tested for the presence of microorganisms or nucleic acids thereof.

13. The device of claim 10, wherein said plurality of loci are wells of a multi-well plate.

14. A kit for differentiating between bacteria, wherein said kit comprises the device of claim 10.

15. The kit of claim 14, wherein said kit comprises one or more of, a specimen lysis reagent;

a probe hybridization reagent;

a selection reagent;

an alkaline hydrolysis reagent;

or an internal control nucleic acid.