Genus, group, species and/or strain specific 16S rDNA sequences

• US 20060046246A1
• Filed: 04/26/2004
• Published: 03/02/2006
• Est. Priority Date: 04/24/2003
• Status: Abandoned Application

• Current Assignee: Biomerieux Incorporated (Compagnie Merieux Alliance SAS), Oscient Pharmaceuticals Corporation
• Original Assignee: Oscient Pharmaceuticals Corporation
• Inventors: Zeng, Qiandong, Moir, Donald T., Chatellier, Sonia, LaCroix, Bruno, Childress, Darrell
• Application Number:
  US10/831,286
• Publication Number:
  US 20060046246A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

Materials and methods for identifying unique sites in bacterial 16S and 23S rDNA are provided, as well as specific unique sequences of 16S rDNA in select bacteria. The distinguishing moieties will enable rapid differentiation between families, genera, groups, species, strains, subspecies, and isolates of microorganisms. Such differentiation can be performed by using rapid screening kits in combination with in silico analysis for diagnostic, prognastic, epidemiologic, phylogenetic, and other purposes.

Claims

1. A plurality of 16S polynucleotides immobilized to a solid support, wherein the plurality of 16S polynucleotides are subsequences of 16S rDNA and each 16S polynucleotide individually comprises at least one distinguishing moiety, which differentiates between microorganisms by genus, group, species, strain, and/or isolate.

2. The plurality of 16S polynucleotides of claim 1, wherein the 16S polynucleotide is an oligomer of 11 to 45 nucleotides.

3. The plurality of 16S polynucleotides of claim 2, wherein the 16S polynucleotide is an oligomer of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

4. The plurality of 16S polynucleotides of claim 3, wherein the solid support is a bead, slide, chip, microtube, or plate.

5. The plurality of 16S polynucleotides of claim 4, wherein the bead is glass or plastic.

6. The plurality of 16S polynucleotides of claim 1, wherein at least 5 to 1×

10⁶ 16S polynucleotides are immobilized to the solid support.

7. The plurality of 16S polynucleotides of claim 1, wherein at least 5 to 100 16S polynucleotides are immobilized to the solid support.

8. A method of detecting the presence of a microorganism and determining an isolate, a strain, a species, a group, or a genus of a microorganism in a sample suspected of containing the microorganism comprising the steps of:

(A) selecting at least one primer pair to amplify at least a portion of a 16S rDNA of the sample;

(B) amplifying the 16S rDNA of the sample with the at least one primer pair;

(C) contacting the amplified rDNA with at least one isolated nucleic acid comprising at least one distinguishing moiety;

(D) incubating the amplified rDNA and the isolated nucleic acid under hybridizing conditions which allow hybridization in a sequence-specific manner between the sample and the at least one isolated nucleic acid to form a hybridization product;

(E) detecting the hybridization product and thereby one or more distinguishing moieties of the microorganism; and

(F) determining the isolate, strain, species, group, and/or genus of the microorganism by the presence of the one or more distinguishing moieties.

9. The method of claim 8, wherein the sample is a food, a biological sample taken from a subject, an environmental sample, or a plant.

10. The method of claim 9, wherein the subject is an agricultural animal or a mammal.

11. A kit for the detection and identification of at least one microorganism by genus, group, species, strain, and/or isolate in a sample comprising:

(A) at least one primer pair for amplification of at least a portion of a 16S rRNA of the microorganism;

(B) two or more nucleic acids comprising at least two critical residues of a 16S rDNA which distinguish the microorganism by genus, group, species, strain, or isolate;

(C) a hybridization buffer to allow sequence-specific hybridization between the probes and the nucleic acids present in the sample, or to allow sequence-specific hybridization between the probes and the nucleic acids of amplified products of the sample; and

(D) a detection moiety.

12. The kit of claim 11, wherein the kit further comprises a detection means, instructions for use of the kit, a wash buffer, and/or a hybridization buffer or any combination thereof.

13. The kit of claim 11, wherein the sample is a plant sample, an environmental sample, a food, or a biological sample obtained from a subject.

14. The kit of claim 11, wherein the 16S rDNA distinguishing moiety is a distinguishing moiety of Table 20 and/or Table 21.

28. A method of diagnosing a subject and determining the microorganism causing an infection in the subject comprising the steps of:

(A) obtaining a sample from the subject; and

(B) screening the sample for the microorganism using the kit of claim 11.

15. A composition comprising a plurality of probes of Table 20 and/or Table 21, wherein each probe comprises at least one distinguishing moiety, and wherein the plurality of probes are immobilized on a substrate.

16. The composition of claim 15, wherein the substrate is a bead, a microarray plate, or a microarray slide.

17. The composition of claim 16, wherein the bead is glass or plastic.

18. The composition of claim 16, wherein the composition comprises a plurality of beads forming a matrix and the matrix is in the form of an affinity column.

19. The composition of claim 15, wherein the plurality of distinguishing moieties are genus specific, group specific, species specific, strain specific, isolate specific, or any combination thereof.

20. The composition of claim 15 further comprising a linker bound to each probe.

21. The composition of claim 15, wherein the plurality of probes are individually at least 15 nucleotides to 30 nucleotides.

22. A database comprising a plurality of distinguishing moieties which differentiate a microorganism by genus, group, species, strain, or isolate, and wherein the database comprises at least two distinguishing moieties from Tables 2, 20, or 21.

23. The database of claim 22, wherein the database further differentiates the microorganism by genus and species.

24. A reference database comprising a plurality of distinguishing moieties, wherein the distinguishing moieties include distinguishing moieties of Table 20 and/or Table 21 in relational form with a means for querying the reference database.

25. A computerized storage and retrieval system of critical residues comprising:

a data entry means;

a display means;

a programmable central processing unit; and

a data storage means having 16S rRNA distinguishing moieties and annotated information on attributes of the 16S rRNA distinguishing moieties electronically stored in a relational database.

26. The computerized storage and retrieval system of claim 25, wherein the stored 16S rRNA critical residues are selected from Tables 2, 20, and 21.

27. A method of identifying distinguishing moieties in a 16S bacterial rRNA or rDNA comprising the steps of:

(A) obtaining a nucleotide sequence of a genetic locus shared by two or more different bacterial strains, species, or genera;

(B) dividing the nucleotide sequence into a set of oligomers of length “

n”

which overlap by “

x”

nucleotides, wherein “

x”

is at least one nucleotide less than “

n” and

wherein said overlapping oligomers span the length of the sequence of the genetic locus;

(C) comparing an oligomer using a comparative algorithm against at least one database of nucleotide sequences for that locus from a plurality of bacterial strains, species, or genera, wherein the nucleotide sequences are stored in at least one database; and

(D) determining whether the oligomer has a nucleotide sequence which matches, or has no more than one mismatch with, a portion of all available nucleotide sequences for the locus of the strain, species, or genus of origin, or whether the nucleotide sequence has at least two mismatches when aligned with any other strain, species, or genus, wherein the at least two mismatches when aligned correspond to distinguishing moieties which differentiate between strain, species or genus.