Compositions and methods for detecting streptococcus agalactiae surface immunogenic protein genes

• US 20040009482A1
• Filed: 07/09/2002
• Published: 01/15/2004
• Est. Priority Date: 07/09/2002
• Status: Abandoned Application

• Current Assignee: Applied Gene Technologies Incorporated
• Original Assignee: Applied Gene Technologies Incorporated
• Inventors: Dattagupta, Nanibhushan, Shah, Ketan
• Application Number:
  US10/192,278
• Publication Number:
  US 20040009482A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

This invention relates generally to Group B streptococci (GBS) Streptococcus agalactiae detection. More specifically, the present invention provides for novel probes for a specific and sensitive diagnostic test of GBS. These GBS-specific probes hybridize with the the surface immunogenic protein (sip) gene. Arrays comprising the probes immobilized on a support for hybridization analysis and methods for GBS detection using the probes are also provided.

Claims

1. An oligonucleotide probe for detecting Streptococcus agalactia in a sample comprising a nucleotide sequence having at least 90% identity with a sequnce selected from the group consisting of:

2. The oligonucleotide probe of claim 1, further comprising a nucleotide sequence having 100% identity with a sequence selected from the group consisting of:

3. The probe of claim 1, which comprises DNA, RNA, PNA or a derivative thereof.

4. The probe of claim 1, which comprises both DNA and RNA or derivatives thereof.

5. The probe of claim 1, which is labeled.

6. The probe of claim 5, wherein the label is selected from the group consisting of a chemical, an enzymatic, an immunogenic, a radioactive, a fluorescent, a luminescent and a FRET label.

7. An array of oligonucleotide probes immobilized on a support for detecting Streptococcus agalactia, which array comprises a support suitable for use in nucleic acid hybridization having immobilized thereon a plurality of oligonucleotide probes, at least one of said probes being a probe according to claim 1.

8. The array of claim 7, wherein the plurality of probes comprise DNA, RNA, PNA or a derivative thereof.

9. The array of claim 7, wherein at least one of the probes comprises both DNA and RNA or derivatives thereof.

10. An array of oligonucleotide probes immobilized on a support for detecting Streptococcus agalactia, which array comprises a support suitable for use in nucleic acid hybridization having immobilized thereon a plurality of oligonucleotide probes, at least one of said probes being a probe according to claim 2.

11. The array of claim 7, wherein at least one of the probes is labeled.

12. The array of claim 11, wherein the label is selected from the group consisting of a chemical, an enzymatic, an immunogenic, a radioactive, a fluorescent, a luminescent and a FRET label.

13. The array of claim 7, wherein the support comprises a surface that is selected from the group consisting of a silicon, a plastic, a glass, a ceramic, a rubber, and a polymer surface.

14. A method for detecting Streptococcus agalactia in a sample, comprising the steps of:

a) providing an oligonucleotide probe comprising a nucleotide sequence, or a complementary strand thereof, having at least 90% identity with a sequence selected from the group consisting of;

5′

-ATCGACAATGGCAGCTTCGC-3′

,(SEQ ID NO;

6) 5′

-CTGGTCAAACAACAGCTACTG-3′

,(SEQ ID NO;

7) 5′

-AACAGGTATCACCAGCTCCT-3′

,(SEQ ID NO;

8) 5′

-GAACAGGTATCACCAGCTCCTG-3′

,(SEQ ID NO;

9) 5′

-GCAAGTGTTAAAGTAGTCACTC-3′

,(SEQ ID NO;

10) 5′

-GGCAAGTGTTAAAGTAGTCACTCC-3′

,(SEQ ID NO;

11) 3′

-TAGCTGTTACCGTCGAAGCG-5′

,(SEQ ID NO;

12) 3′

-GACCAGTTTGTTGTCGATGAC-5′

,(SEQ ID NO;

13) 3′

-TTGTCCATAGTGGTCGAGGT-5′

,(SEQ ID NO;

14) 3′

-CTTGTCCATAGTCCTCGAGGAC-5′

,(SEQ ID NO;

15) 3′

-CGTTCACAATTTCATCAGTGAG-5′

,(SEQ ID NO;

16)and 3′

-CCGTTCACAATTTCATCTGTGAGG-5′

;

(SEQ ID NO;

17)

b) contacting said probe with a sample containing or suspected of containing a Streptococcus agalactia target nucleotide sequence under conditions suitable for hybridization between said probe and said target nucleotide sequence; and

c) assessing hybridization between said probe and said target nucleotide sequence to detect said Streptococcus agalactia in said sample.

15. The method of claim 14, wherein the probe comprises DNA, RNA, PNA or a derivative thereof.

16. The method of claim 14, wherein the probe comprises both DNA and RNA or derivatives thereof

17. The method of claim 14, wherein the probe comprises a nucleotide sequence that is selected from the group consisting of:

18. The method of claim 14, wherein the probe or the Streptococcus agalactiae target nucleotide sequence is labeled.

19. The method of claim 18, wherein the label is selected from the group consisting of a chemical, an enzymatic, an immunogenic, a radioactive, a fluorescent, a luminescent and a FRET label.

20. The method of claim 14, wherein the probe or the Streptococcus agalactiae target nucleotide sequence is immobilized on a support.

21. The method of claim 14, wherein a plurality of the probes immobilized on a support is used.

22. The method of claim 14, wherein a plurality of samples is assayed.

23. The method of claim 22, wherein the plurality of samples is assayed simultaneously.

24. The method of claim 14, wherein a sample of human origin is assayed.

25. The method of claim 14, wherein the sample is selected from the group consisting of sputum, urine, blood, tissue section, food, soil and water sample.

26. The method of claim 14, wherein the Streptococcus agalactia strain is selected from the group consisting of:

NCS215, NCS915, NCS535, NCS246, COH1 , C388/90, CNTC 1/82, CNCTC 1/82, and NT6.

27. A method for detecting Streptococcus agalactia in a sample comprising the steps of:

a) providing an oligonucleotide probe comprising a nucleotide sequence that hybridizes with a target nucleotide sequence, wherein the target nucleotide sequence is all or part of the sip gene, or a complementary strand thereof, and wherein the probe has a G+C content from about 30 to 70%, a Tm value from about 40 to 80°

C., a length of at least 8 nucleotides and does not contain any hairpin secondary structure;

b) contacting said, probe with a sample containing or suspected of containing a Streptococcus agalactia target nucleotide sequence under conditions suitable for hybridization between said probe and said target nucleotide sequence; and

c) assessing hybridization between said probe and said target nucleotide sequence to detect said Streptococcus agalactia in said sample.

28. A method for detecting Streptococcus agalactia in a sample comprising the steps of:

a) providing an oligonucleotide probe comprising a nucleotide sequence that hybridizes under high stringency with a target comprising a nucleotide sequence selected from the group consisting of;

5′

-ATCGACAATGGCAGCTTCGC-3′

,(SEQ ID NO;

6) 5′

-CTGGTCAAACAACAGCTACTG-3′

,(SEQ ID NO;

7) 5′

-AACAGGTATCACCAGCTCCT-3′

,(SEQ ID NO;

8) 5′

-GAACAGGTATCACCAGCTCCTG-3′

,(SEQ ID NO;

9) 5′

-GCAAGTGTTAAAGTAGTCACTC-3′

,(SEQ ID NO;

10) 5′

-GGCAAGTGTTAAAGTAGTCACTCC-3′

,(SEQ ID NO;

11) 5′

-TAGCTGTTACCGTCGAAGCG-3′

,(SEQ ID NO;

12) 3′

-TAGCTGTTACCGTCGAAGCG-5′

,(SEQ ID NO;

12) 3′

-GACCAGTTTGTTGTCGATGAC-5′

,(SEQ ID NO;

13) 3′

-TTGTCCATAGTGGTCGAGGT-5′

,(SEQ ID NO;

14) 3′

-CTTGTCCATAGTCCTCGAGGAC-5′

,(SEQ ID NO;

15) 3′

-CGTTCACAATTTCATCAGTGAG-5′

,(SEQ ID NO;

16)and 3′

-CCGTTCACAATTTCATCTGTGAGG-5′

;

(SEQ ID NO;

17)

b) contacting said probe with a sample containing or suspected of containing a Streptococcus agalactia target nucleotide sequence under conditions suitable for hybridization between said probe and said target nucleotide sequence; and

c) assessing hybridization between said probe and said target nucleotide sequence to detect said Streptococcus agalactia in said sample.

29. An oligonucleotide probe for detecting Streptococcus agalactia in a sample comprising a nucleotide sequence that hybridizes with a target nucleotide sequence, or a complementary strand thereof, selected from the group consisting of:

30. The probe of claim 29, which comprises DNA, RNA, PNA or a derivative thereof.

31. The probe of claim 29, which comprises both DNA and RNA or derivatives thereof.

32. The probe of claim 29, which is labeled.

33. The probe of claim 32, wherein the label is selected from the group consisting of a chemical, an enzymatic, an immunogenic, a radioactive, a fluorescent, a luminescent and a FRET label.