Method for rapid and accurate identification of microorganisms

• US 20030134293A1
• Filed: 05/16/2002
• Published: 07/17/2003
• Est. Priority Date: 11/16/1999
• Status: Abandoned Application

• Current Assignee: Trovagene, Inc.
• Original Assignee: Trovagene, Inc.
• Inventors: Liu, Zhiping
• Application Number:
  US10/151,484
• Publication Number:
  US 20030134293A1
• 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 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Methods and compositions useful for rapid identification of microorganisms are provided. Methods and compositions useful for rapid and simultaneous identification in a biological sample of multiple microorganisms, including bacteria, yeast, fungi and viruses are also provided. The methods and compositions utilize amplification techniques and sequence specific hybridization to detect species specific polynucleotide sequence in a sample. Novel methods for coupling oligonucleotide probes to glass surfaces with increased efficiency are also provided.

Claims

1. A method of identifying an organism among a population of organisms in a biological sample, the method comprising:

obtaining genetic material from the sample;

contacting the genetic material with at least a first primer and at least a related second primer corresponding to a pair of conserved regions in the genome of the population of organisms, wherein the first primer hybridizes upstream and the second primer hybridizes downstream of a target sequence in the genetic material in the sample, and further wherein the target sequence is less conserved than the primer binding sequences and is characteristic of the organism;

amplifying the target sequence;

contacting a solid support comprising a probe substantially complementary to the target sequence with the amplified target sequence; and

detecting hybridization of the target sequence to the probe, wherein hybridization is indicative of the presence of the organism in the sample.

2. The method of claim 1, further comprising diagnosing a disease or disorder associated with an organism, wherein hybridization of the target sequence to the probe is indicative of the presence of the organism in the sample and correlating the organism to the disease or disorder.

3. The method of claim 1, wherein the organism is selected from the group consisting of a prokaryotic organism, viral organism or a single cell eukaryotic organism.

4. The method of claim 1, wherein the prokaryotic organism is a gram positive or gram negative bacteria.

5. The method of claim 1, wherein the biological sample is a fluid sample.

6. The method of claim 5, wherein the fluid sample is blood, urine, cerebrospinal fluid, sputum, tracheal aspirate or pleural fluid.

7. The method of claim 1, wherein the biological sample is a tissue sample.

8. The method of claim 1, wherein the genetic material is DNA or RNA.

9. The method of claim 1, wherein the primer is an oligomer of DNA, RNA, or PNA.

10. The method of claim 1, wherein the target sequence is species specific.

11. The method of claim 1, wherein the target sequence is amplified by PCR.

12. The method of claim 10, wherein the probe is complementary to the species specific target sequence.

13. The method of claim 1, wherein the solid support is selected from the group consisting of nylon, glass, silicon, polymer, plastic, ceramics, metal or optical fiber.

14. The method of claim 1, wherein the solid support is a biochip.

15. The method of claim 1, wherein the detection is by measuring radioactivity, fluorescence or chemiluminescence.

16. An array of oligonucleotide probes immobilized on a solid support, the array comprising:

a plurality of probes having a sequence corresponding to a species specific polynucleotide target sequence wherein the species specific target sequence is flanked on either side by oligonucleotide sequences that are conserved across a plurality of organisms.

17. The array of claim 16, wherein the plurality of organisms are of the same family.

18. The array of claim 16, wherein the plurality of organisms are of the same genus.

19. The array of claim 16, wherein the plurality of organisms correlate with at least one common disease or disorder.

20. A kit comprising:

at least one oligonucleotide primer complementary to a conserved region of genetic material in a population of organisms; and

a solid support having attached thereto a species-specific probe capable of hybridizing to a target sequence, the target sequence flanked by the at least one primer.

21. A method for identifying at least two organisms from a population of organisms in a biological sample, comprising:

obtaining genetic material from the biological sample;

contacting the genetic material with at least a first primer and at least a related second primer corresponding to a pair of conserved regions in the genome of the population of organisms, wherein the first primer hybridizes upstream and the second primer hybridizes downstream of a target sequence in the genetic material in the sample, and further wherein the target sequence is less conserved than the primer binding sequences and each target sequence is characteristic of one of the at least two organisms;

amplifying the target sequence;

providing a solid support comprising at least two probes selected from the at least two different organisms, wherein the at least two probes comprise sequences that are substantially complementary to the target sequence in the organism from which the probe sequences were selected;

contacting the solid support with amplification products of the amplified target sequence; and

detecting hybridization of the target sequence to the probe, wherein hybridization to a probe is indicative of the presence of the corresponding organism in the sample.

22. The method of claim 21, wherein the at least two different organisms are selected from the group consisting of bacteria, yeast, paramecia, trypanosoma, unicellular eukaryotes, and viruses.

23. The method of claim 21, wherein the target sequence comprises RecA or FtsY or both.

24. A method of distinguishing a presence of at least two organisms from a population of organisms in a biological sample, comprising:

obtaining genetic material from the biological sample;

contacting the genetic material with at least a first primer and at least a related second primer corresponding to a pair of conserved regions in the genome of the population of organisms, wherein the first primer hybridizes upstream and the second primer hybridizes downstream of a target sequence in the genetic material in the sample, and further wherein the target sequence is less conserved than the primer binding sequences and each target sequence is characteristic of one of the at least two organisms;

amplifying the target sequence;

providing a solid support comprising at least two probes selected from the at least two different organisms, wherein the at least two probes comprise sequences that are substantially complementary to the target sequence and differentially hybridize to the target sequence depending on a hybridization condition;

contacting the solid support with amplification products of the amplified target sequence under a hybridization condition wherein hybridization to a probe corresponding to any one of the at least two organisms is preferred; and

detecting hybridization of the target sequence to the probe corresponding to any one of the at least two organisms, wherein hybridization to the probe is indicative of the presence of the corresponding organism in the sample.

25. The method of claim 24, wherein the hybridization condition comprises stringency or temperature or both.

26. The method of claim 24, wherein the at least two different organisms are selected from the group consisting of bacteria, yeast, paramecia, trypanosoma, unicellular eukaryotes, and viruses.

27. The method of claim 24, wherein the target sequence comprises RecA or FtsY or both.

28. A method of identifying a target sequence in a biological sample, comprising:

obtaining genetic material from the biological sample;

contacting the genetic material with at least a first primer and at least a related second primer corresponding to a pair of conserved regions in the genome of a population of organisms, wherein the first primer hybridizes upstream and the second primer hybridizes downstream of a target sequence in the genetic material in the sample, and further wherein the target sequence is less conserved than the primer binding sequences;

amplifying the target sequence; and

determining the sequence of amplification products of the amplified target sequence.

29. The method of claim 28, further comprising:

identifying an organism associated with the sequenced target sequence by comparing the sequence of the amplified target with a known sequence of the corresponding target in the organism.

30. The method of claim 29, wherein the organism comprises a bacteria, a yeast, an unicellular eukaryote or a virus.

31. The method of claim 29, wherein the target sequence comprises RecA or FtsY or both.

32. A probe corresponding to a RecA gene wherein the probe is selected from the group consisting of polynucleotides having SEQ ID NOS 53-80.

33. A probe corresponding to a FtsY gene wherein the probe is selected from the group consisting of polynucleotides having SEQ ID NOS 81-104.

34. A probe corresponding to a human RSV virus wherein the probe is selected from the group consisting of polynucleotides having SEQ ID NOS 107 and 108.

35. A primer oligonucleotide for use as a forward PCR primer for an amplification of FtsY sequences in an organism, wherein the oligonucleotide is selected from the group consisting of:

(a) an oligonucleotide wherein five nucleotides at a 3′

end of the oligonucleotide bears at least about 80% sequence identity to five nucleotides at a 3′

end of oligonucleotides selected from the group consisting of SEQ ID NOS 1-14; and

(b) an oligonucleotide wherein the oligonucleotide bears at least about 70% sequence identity to oligonucleotides selected from the group consisting of SEQ ID NOS 1-14.

36. A primer oligonucleotide for use as a reverse PCR primer for use with a related primer of claim 35, for an amplification of FtsY sequences in an organism, wherein the oligonucleotide is selected from the group consisting of:

(a) an oligonucleotide wherein wherein five nucleotides at a 3′

end of the oligonucleotide bears at least about 80% sequence identity to five nucleotides at a 3′

end of the oligonucleotides selected from the group consisting of SEQ ID NOS 15-27; and

(b) an oligonucleotide wherein the oligonucleotide bears at least about 70% sequence identity to oligonucleotides selected from the group consisting of SEQ ID NOS 15-27.

37. A primer oligonucleotide for use as a forward PCR primer for an amplification of RecA sequences in an organism, wherein the oligonucleotide is selected from the group consisting of:

(a) an oligonucleotide wherein five nucleotides at a 3′

end of the oligonucleotide bears at least about 80% sequence identity to five nucleotides at a 3′

end of oligonucleotides selected from the group consisting of SEQ ID NOS 28-40; and

(b) an oligonucleotide wherein the oligonucleotide bears at least about 70% sequence identity to oligonucleotides selected from the group consisting of SEQ ID NOS 28-40.

38. A primer oligonucleotide for use as a reverse PCR primer for use with a related primer of claim 37, for an amplification of RecA sequences in an organism, wherein the oligonucleotide is selected from the group consisting of:

(a) an oligonucleotide wherein five nucleotides at a 3′

end of the oligonucleotide bears at least about 80% sequence identity to five nucleotides at a 3′

end of oligonucleotides selected from the group consisting of SEQ ID NOS 41-52; and

(b) an oligonucleotide wherein the oligonucleotide bears at least about 70% sequence identity to oligonucleotides selected from the group consisting of SEQ ID NOS 41-52.

39. A primer oligonucleotide for use as a forward PCR primer for an amplification of a human RSV sequence, wherein the oligonucleotide is selected from the group consisting of:

(a) an oligonucleotide wherein five nucleotides at a 3′

end of the oligonucleotide bears at least about 80% sequence identity to five nucleotides at a 3′

end of oligonucleotides selected from the group consisting of SEQ ID NO 105; and

(b) an oligonucleotide wherein the oligonucleotide bears at least about 70% sequence identity to oligonucleotides selected from the group consisting of SEQ ID NO 105.

40. A primer oligonucleotide for use as a reverse PCR primer for use with a related primer of claim 39, for an amplification of a human RSV sequence, wherein the oligonucleotide is selected from the group consisting of:

(a) an oligonucleotide wherein five nucleotides at a 3′

end of the oligonucleotide bears at least about 80% sequence identity to five nucleotides at a 3′

end of oligonucleotides selected from the group consisting of SEQ ID NO 106; and

(b) an oligonucleotide wherein the oligonucleotide bears at least about 70% sequence identity to oligonucleotides selected from the group consisting of SEQ ID NO 106.

41. A polynucleotide comprising SEQ ID NO 109, wherein the polynucleotide corresponds to a FtsY gene of Bacteroides fragilis.

42. A hybridization probe for use in a detection of a FtsY gene comprising a selectively hybridizable segment of the polynucleotide of claim 41.

43. A PCR primer oligonucleotide sequence for use in detection of a FtsY gene, wherein the PCR primer oligonucleotide sequence comprises at least five continuous nucleotides of a polynucleotide comprising SEQ ID NO 109.

44. A method for increasing the efficiency of coupling of an oligonucleotide to a solid substrate, the method comprising:

applying a positive electrostatic potential to a surface of the solid substrate, whereby the positive electrostatic potential increases a concentration of oligonucleotides and negatively charged molecules to the surface of the solid substrate.

45. A method for increasing the efficiency of coupling of an oligonucleotide to a glass substrate by forming an Epoxy derivative of a surface of the glass substrate, the method comprising:

applying an epoxy derivative to the surface of the glass substrate.

46. The method of claim 45, wherein the epoxy comprises a three member ring.

47. An array for testing a biological fluid for the presence of at least one organism correlated with at least one condition selected from meningitis and encephalitis, the array comprising:

at least two probes comprising sequences of clusters of orthologous groups (COGs) selected from at least one of FtsY (COG0552), 3-phosphoglycerate kinase (COG0060), and isoleucyl-tRNA synthetase (COG0126).

48. The array of claim 47 wherein the organism is selected from the group consisting of Herpes Simplex Virus 1, Herpes Simplex Virus 2, Streptococcus pneumoniae, Streptococcus agalactiae (group B), Neisseria meningitidis, Hemophilus influenzae, and Listeria monocytogenes.

49. A method for selecting one or more COGs for use in an array for detecting at least one organism related to a pathologic condition, the method comprising:

examining a sequence of at least one COG from an organism related to the pathologic condition; and

selecting one or more COGs wherein the selected COGs satisfy a criteria comprising

(i) the COG is highly conserved in bacterium, eukaryote, and archeabacterium;

(ii) the COG is present in a single-copy in a genome; and

(iii) the COG comprises at least two highly conserved domains flanking a more divergent domain of the coding sequence greater than 200 base pairs in length.

50. The method of claim 49 wherein the COG is selected from the group consisting of COG0173 (aspartyl-tRNA synthetase), COG0525 (valyl-tRNA synthetase), and COG0112 (glycine hydroxymethyl transferase).

51. The method of claim 49 wherein the COG is selected from a COG listed in Table 4.