PROBES AND METHODS FOR DETECTION OF PATHOGENS AND ANTIBIOTIC RESISTANCE

• US 20080199863A1
• Filed: 05/01/2007
• Published: 08/21/2008
• Est. Priority Date: 11/01/2004
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California), U.S. Government Represented By The Department Of Veterans Affairs
• Original Assignee: Regents of the University of California (University of California), U.S. Government Represented By The Department Of Veterans Affairs
• Inventors: Li, Yang, Haake, David A., Liao, Joseph C., Churchill, Bernard M., Suchard, Marc A., Mastali, Mitra
• Granted Patent: US 7,763,426 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

Described are probes and methods for detecting pathogens and antibiotic resistance of a specimen. The method comprises contacting the specimen with a growth medium; and lysing the specimen to release nucleic acid molecules from the specimen. The lysate of the specimen is contacted with a capture probe immobilized on a substrate, wherein the capture probe comprises an oligonucleotide that specifically hybridizes with a first target nucleic acid sequence region of ribosomal RNA. The lysate is in contact with a detector probe that comprises a detectably labeled oligonucleotide that specifically hybridizes with a second target nucleic acid sequence region of ribosomal RNA. The presence or absence of labeled oligonucleotide complexed with the substrate is determined. Detection of labeled oligonucleotide complexed with the substrate is indicative of the presence of pathogen. Performing the method in the presence and absence of an antibiotic permits detection of antibiotic resistance.

Claims

1. A pair of oligonucleotide probes, comprising a capture probe and a detector probe, each of which is 10-35 bases in length, and that specifically hybridize under highly stringent conditions to a target capture region and a target detector region, respectively, of bacterial ribosomal RNA (rRNA), wherein the target capture region and target detector region are, or are fully complementary to, nucleic acid molecules corresponding to, a pair of sequences selected from the group consisting of:

(a) SEQ ID NO;

22, target capture region of P. mirabilis, and

SEQ ID NO;

23, target detector region of P. mirabilis,

(b) SEQ ID NO;

24, target capture region of E. faecalis, and

SEQ ID NO;

25, target detector region of E. faecalis,

(c) SEQ ID NO;

26, target capture region of K. pneumoniae, and

SEQ ID NO;

27, target detector region of K. pneumoniae, and

(d) SEQ ID NO;

28, target capture region of E. coli, and

SEQ ID NO;

29, target detector region of E. coli,

(e) SEQ ID NO;

30, target capture region of Enterobacteriaceae, and

SEQ ID NO;

31, target detector region of Enterobacteriaceae;

(f) SEQ ID NO;

32, target capture region of P. aeruginosa, and

SEQ ID NO;

33, target detector region of P. aeruginosa;

(g) SEQ ID NO;

34, target capture region of Acinetobacter baumani, and

SEQ ID NO;

35, target detector region of Acinetobacter baumani;

(h) SEQ ID NO;

36, target capture region of Enterobacter aerogenes, and

SEQ ID NO;

37, target detector region of Enterobacter aerogenes;

(i) SEQ ID NO;

38, target capture region of Serratia marcescens, and

SEQ ID NO;

39, target detector region of Serratia marcescens;

(j) SEQ ID NO;

40, target capture region of Stenotrophomonas maltophilia, and

SEQ ID NO;

41, target detector region of Stenotrophomonas maltophilia; and

(k) SEQ ID NO;

42, universal bacterial target capture region, and

SEQ ID NO;

43, universal bacterial target detector region.

2. The probe pair of claim 1 that is a pair of nucleic acid sequences selected from the group consisting of:

(a) Escherichia coli capture probe, SEQ ID NO;

44; and

detector probe, SEQ ID NO;

45;

(b) Proteus mirabilis, capture probe, SEQ ID NO, 46; and

detector probe, SEQ ID NO, 47;

(c) Klebsiella pneumoniae capture probe, SEQ ID NO;

48; and

detector probe, SEQ ID NO;

49;

(d) Pseudomonas aeruginosa capture probe, SEQ ID NO. 50; and

detector probe, SEQ ID NO;

51;

(e) Enterococcus spp. capture probe, SEQ ID NO;

52, and detector probe, SEQ ID NO;

53;

(f) Enterobacteriaceae capture probe, SEQ ID NO;

54; and

detector probe. SEQ ID NO;

55;

(g) Acinetobacter baumani capture probe, SEQ ID NO;

56; and

detector probe, SEQ ID NO;

57;

(h) Enterobacter aerogenes capture probe, SEQ ID NO;

58, and detector probe, SEQ ID NO;

59;

(i) Serratia marcescens capture probe, SEQ ID NO;

60; and

detector probe, SEQ ID NO;

61;

(j) Stenotrophomonas maltophilia capture probe. SEQ ID NO;

62; and

detector probe, SEQ ID NO;

63; and

(k) universal bacterial capture probe, SEQ ID NO;

64; and

detector probe, SEQ ID NO;

65.

3. The probe pair of claim 1, wherein the detector probe is labeled with a detectable marker.

4. A method for detecting the presence of a pathogen in a specimen, the method comprising:

(a) contacting a lysate of the specimen with a capture probe immobilized on a substrate, wherein the capture probe comprises an oligonucleotide that specifically hybridizes with a first target nucleic acid sequence region of the pathogen, wherein the first target nucleic acid sequence region is selected from SEQ ID NOS.;

22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and 42, and wherein the contacting occurs under conditions that permit hybridization of the capture probe with the first target nucleic acid sequence;

wherein the lysate is in contact with a detector probe that comprises a detectably labeled oligonucleotide that specifically hybridizes with a second target nucleic acid sequence region of the pathogen, wherein the target nucleic acid sequence region is selected from SEQ ID NOS;

23, 25, 27, 29, 31, 33, 36, 37, 39, 41 and 43,

(b) determining the presence of the detector probe, whereby detection of the detector probe complexed with the substrate is indicative of the presence of the corresponding pathogen.

5. The method of claim 4, wherein the conditions that permit hybridization are a temperature of 20°

C. to 39°

C. and a buffered saline solution.

6. The method of claim 5, wherein the conditions that permit hybridization are a temperature of 20°

C. to 25°

C. and a buffered saline solution.

7. The method of claim 4, wherein the lysate is brought into contact with the detector probe prior to the contacting of the lysate with the capture probe.

8. The method of claim 4, wherein the lysate is brought into contact with the detector probe after the contacting of the lysate with the capture probe.

9. The method of claim 4, wherein the lysate is prepared by contacting the specimen with a first lysis buffer comprising a non-denaturing detergent and lysozyme.

10. The method of claim 9, wherein the lysate is further prepared by contacting the specimen with a second lysis buffer comprising NaOH.

11. The method of claim 10, wherein the contacting of the specimen with the second lysis buffer occurs prior to the contacting of the specimen with the first lysis buffer.

12. The method of claim 10, wherein the contacting of the specimen with second lysis buffer occurs after the contacting of the specimen with the first lysis buffer.

13. The method of claim 10, wherein the contacting of the specimen with the first and/or second lysis buffers occurs at 20°

C. to 39°

C.

14. The method of claim 10, wherein the contacting of the specimen with the first and/or second lysis buffers occurs for about 5 minutes per lysis buffer.

15. The method of claim 4, wherein the detectable label is at the 3 end of the detector probe.

16. The method of claim 4, wherein the oligonucleotide probes are 10-35 bases in length.

17. The method of claim 4, wherein the oligonucleotide probes are 10-25 bases in length.

18. The method of claim 4, wherein the determining comprises an electrochemical sensor assay and the presence of detector probe is determined by measuring current output.

19. The method of claim 18, wherein the determining comprises comparing current output at 15 minutes after contacting the specimen with the growth medium.

20. The method of claim 4, wherein the specimen is a bodily fluid.

21. The method of claim 20, wherein the specimen is urine.

22. A method for detecting antibiotic susceptibility of a specimen, the method comprising:

(a) detecting the presence of a pathogen in the specimen according to the method of claim 4;

(b) quantifying the pathogen detected in step (a);

(c) contacting the lysate of the specimen with a capture probe immobilized on a substrate, wherein the capture probe specifically hybridizes with a first target nucleic acid sequence region of the detected pathogen'"'"'s ribosomal RNA;

wherein the lysate is in contact with a detector probe that comprises a detectably labeled oligonucleotide that specifically hybridizes with a second target nucleic acid sequence region of the detected pathogen'"'"'s ribosomal RNA; and

(d) inoculating the specimen into a growth medium;

(e) repeating step (d) in the presence of an antibiotic;

(f) comparing the amount of labeled oligonucleotide complexed with the substrate, whereby detection of less labeled oligonucleotide complexed with the substrate in the presence of antibiotic is indicative of susceptibility to the antibiotic.

23. An assay kit for detecting a plurality of pathogens comprising:

(a) an electrochemical sensor array comprising a plurality of electrodes;

(b) a plurality of paired oligonucleotide probes that specifically hybridize to target nucleic acid sequences of bacterial ribosomal RNA, wherein each pair of probes comprises;

(i) a capture probe bound to one of the electrodes, the capture probe having a first detectable label, wherein the target nucleic acid sequence region is selected from SEQ ID NOS;

22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and 42; and

(ii) a detector probe having a second detectable label, wherein the target nucleic acid sequence region is selected from SEQ ID NOS, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43;

wherein the capture probe and detector probe of each pair of probes specifically hybridize to adjacent nucleic acid sequences of bacterial ribosomal RNA.

24. The assay kit of claim 23, wherein the second detectable label is at the 3′

end of the detector probe.

25. The assay kit of claim 23, wherein the oligonucleotide probes are 10-35 bases in length.

26. The assay kit of claim 23, wherein the oligonucleotide probes are 10-25 bases in length.

27. The assay kit of claim 23, wherein the bacterial ribosomal RNA is 5S, 16S or 23S ribosomal RNA.