Method and kit for identifying antibiotic-resistant microorganisms
View Patent ↗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.
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.