DETECTION METHOD FOR NEISSERIA GONORRHOEAE

• US 20170260572A1
• Filed: 09/17/2015
• Published: 09/14/2017
• Est. Priority Date: 09/17/2014
• Status: Active Grant

• Current Assignee: Binx Health Limited
• Original Assignee: Atlas Genetics Limited
• Inventors: Filer, Danny, Ferrao, Claire, Chadwick, Sharon
• Granted Patent: US 10,519,514 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

A method of detecting the presence of Neisseria gonorrhoeae in a sample. The method involves detecting a first target sequence taken from the NGO1642 gene and/or a second target sequence taken from the NGO1012 gene. The method may involve a step of amplifying the target sequence, and may involve hybridising the target sequence to a nucleic acid probe and identifying hybridisation. The method may involve simultaneous detection of other target sequences, e.g. from other pathogens.

Claims

1. A method of detecting the presence of Neisseria gonorrhoeae in a sample, comprising a step of detecting:

a first target sequence of NGO1642 or a fragment thereof; and

/or a second target sequence of NGO1012 or a fragment thereof.

2. The method of claim 1, wherein said step of detecting comprises hybridising each target sequence to a nucleic acid probe and identifying the occurrence of hybridisation.

3. The method of claim 1 or claim 2, wherein the first target sequence comprises at least 10 contiguous nucleotides contained in SEQ ID NO:

1 or its complement;

at least 10 nucleotides contained in SEQ ID NO;

25 or its complement;

or at least 10 nucleotides contained in SEQ ID NO;

2 or its complement.

4. The method of claim 1, wherein:

the second target sequence comprises at least 10 contiguous nucleotides contained in SEQ ID NO;

6 or at least 10 nucleotides contained in SEQ ID NO;

7;

the first target sequence comprises at least 30 contiguous nucleotides contained in SEQ ID NO;

25 or SEQ ID NO;

2;

the second target sequence comprises at least 30 contiguous nucleotides contained in SEQ ID NO;

7; and

/or

the detection step comprises detecting both the first target sequence and the second target sequence.

8. The method of claim 2, wherein:

the step of detecting comprises using a first nucleic acid probe comprising a) at least 10 contiguous nucleotides of the nucleotide sequence of SEQ ID NO;

25 or its complement;

wherein the sequence of SEQ ID NO;

25 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

or b) at least 10 contiguous nucleotides of the nucleotide sequence of SEQ ID NO;

2 or its complement;

wherein the sequence of SEQ ID NO;

2 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

the step of detecting comprises using a second nucleic acid probe comprising at least 10 contiguous nucleotides of the nucleotide sequence of SEQ ID NO;

7 or its complement;

wherein the sequence of SEQ ID NO;

7 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

the step of detecting comprises using a first nucleic acid probe comprising or consisting of the nucleotide sequence of SEQ ID NO;

26 or SEQ ID NO;

3; and

/or

the step of detecting comprises using a second nucleic acid probe comprising or consisting of the nucleotide sequence of SEQ ID NO;

8.

12. The method of claim 8, wherein the nucleic acid probe comprises a minor groove binder moiety and/or wherein said nucleic acid probe comprises at least one locked nucleic acid (LNA) nucleotide.

13. The method of claim 1, comprising a step of amplifying the first target sequence and/or the second target sequence using the polymerase chain reaction, transcription mediated amplification, nucleic acid sequence based amplification (NASBA), helicase-dependent amplification, recombinase polymerase amplification, strand displacement amplification or loop-mediated isothermal amplification.

14. The method of claim 13, wherein the step of amplifying the first target sequence and/or the second target sequence uses the polymerase chain reaction.

35. The method of claim 13, wherein said step of amplifying the target sequence involves the use of a forward primer, wherein the forward primer:

comprises a nucleic acid sequence comprising 18 or more contiguous nucleotides selected from SEQ ID NO;

4, wherein the sequence of SEQ ID NO;

4 may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

comprises a nucleic acid sequence comprising 19 or more contiguous nucleotides selected from SEQ ID NO;

9 wherein the sequence of SEQ ID NO;

9 may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

comprises the nucleic acid sequence of SEQ ID NO;

4;

comprises the nucleic acid sequence of SEQ ID NO;

9;

comprises a minor groove binding moiety;

or comprises at least one locked nucleic acid (LNA) nucleotide; and

/or

is conjugated to an electrochemically active label; and

/or

involves the use of a reverse primer, wherein the reverse primer;

comprises a nucleic acid sequence comprising 19 or more contiguous nucleotides selected from SEQ ID NO;

27 or SEQ ID NO;

5;

wherein the sequence of SEQ ID NO;

27 or SEQ ID NO;

5 may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

comprises a nucleic acid sequence comprising 19 or more contiguous nucleotides selected from SEQ ID NO;

10;

wherein the sequence of SEQ ID NO;

10 may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

comprises the nucleic acid sequence of SEQ ID NO;

27 or SEQ ID NO;

5;

comprises the nucleic acid sequence of SEQ ID NO;

10;

comprises a minor groove binding moiety;

or comprises at least one locked nucleic acid (LNA) nucleotide; and

/or

is conjugated to an electrochemically active label.

37. The method of claim 1, wherein said method involves the use of a nucleic acid probe, wherein the nucleic acid probe:

comprises a nucleic acid sequence which comprises 19 or more contiguous nucleotides selected from a) SEQ ID NO;

26 or its complement;

wherein the sequence of SEQ ID NO;

26 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

or b) SEQ ID NO;

3 or its complement;

wherein the sequence of SEQ ID NO;

3 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

is capable of hybridising to the NGO1012 sequence or a fragment thereof;

is capable of hybridising to the NGO1642 sequence or a fragment thereof;

comprises a nucleic acid sequence which comprises 20 or more contiguous nucleotides selected from SEQ ID NO;

8 or its complement;

wherein the sequence of SEQ ID NO;

8 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

comprises a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO;

26 or SEQ ID NO;

3;

consists of the nucleic acid sequence of SEQ ID NO;

26 or SEQ ID NO;

3;

comprises a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO;

8; and

/or

consists of the nucleic acid sequence of SEQ ID NO;

8; and

/or.

38. The method of claim 1, wherein said method further includes a step of detecting the presence of an internal control nucleic acid sequence, optionally wherein:

said internal control nucleic acid sequence is a Pectobacterium atrosepticium nucleic acid sequence; and

/or

said step of detecting the presence of a nucleic acid sequence for use as an internal positive control comprises;

a) a step of amplifying said nucleic acid sequence for use as an internal positive control, and/or b) a step of hybridising the nucleic acid sequence for use as an internal positive control to a probe and a step of identifying the occurrence of hybridisation.

50. The method of claim 1, further comprising detecting the presence of Chlamydia trachomatis in the sample, the method further comprising a step of detecting a Chlamydia trachomatis specific target sequence, optionally wherein the Chlamydia trachomatis specific target sequence comprises at least 10, at least 15, at least 20, at least 25 or at least 30 contiguous nucleotides of SEQ ID NO:

15; and

/or wherein the step of detecting a Chlamydia trachomatis specific target sequence comprises a step of hybridising the Chlamydia trachomatis specific target sequence to a Chlamydia trachomatis specific nucleic acid probe and identifying the occurrence of hybridisation, wherein optionally the Chlamydia trachomatis specific nucleic acid probe comprises at least 10 contiguous nucleotides of SEQ ID NO;

17, optionally further comprising a step of amplifying the Chlamydia trachomatis specific target sequence using PCR, wherein optionally the step of amplifying involves the use of a forward primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO;

18 wherein the sequence of SEQ ID NO;

18 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides and the use of a reverse primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO;

19 wherein the sequence of SEQ ID NO;

19 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

54. The method of claim 1, further comprising detecting the presence of Trichomonas vaginalis in the sample, the method further comprising a step of detecting a Trichomonas vaginalis specific target sequence, optionally:

wherein the Trichomonas vaginalis specific target sequence comprises SEQ ID NO;

20 or a fragment thereof or SEQ ID NO;

21 or a fragment thereof;

wherein the step of detecting comprises a step of hybridising the Trichomonas vaginalis specific target sequence to a Trichomonas vaginalis specific nucleic acid probe and identifying the occurrence of hybridisation, wherein optionally the Trichomonas vaginalis specific nucleic acid probe comprises at least 10 contiguous nucleotides of SEQ ID NO;

22; and

/or

further comprising a step of amplifying the Trichomonas vaginalis specific target sequence using PCR, wherein optionally the step of amplifying involves the use of a forward primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO;

23 wherein the sequence of SEQ ID NO;

23 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides and the use of a reverse primer comprising 12 or more contiguous nucleotides selected from SEQ ID NO;

24 wherein the sequence of SEQ ID NO;

24 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides.

5-7. -7. (canceled)

9-11. -11. (canceled)

15. A composition selected from the group consisting of:

a pair of primers that is capable of amplifying a sequence within NGO1642 to provide an amplicon in a polymerase chain reaction;

a forward primer comprising a nucleic acid sequence comprising 18 or more contiguous nucleotides selected from SEQ ID NO;

4, wherein the sequence of SEQ ID NO;

4 may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

a reverse primer comprising a nucleic acid sequence comprising 19 or more contiguous nucleotides selected from SEQ ID NO;

27 or SEQ ID NO;

5;

wherein the sequence of SEQ ID NO;

27 or SEQ ID NO;

5 may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

a pair of primers that is capable of amplifying a sequence within NGO1012 to provide an amplicon in a polymerase chain reaction;

a forward primer comprising a nucleic acid sequence comprising 19 or more contiguous nucleotides selected from SEQ ID NO;

9 wherein the sequence of SEQ ID NO;

9 may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

a reverse primer comprising a nucleic acid sequence comprising 19 or more contiguous nucleotides selected from SEQ ID NO;

10;

wherein the sequence of SEQ ID NO;

10 may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

a nucleic acid probe comprising a nucleic acid sequence which comprises 19 or more contiguous nucleotides selected from a) SEQ ID NO;

26 or its complement;

wherein the sequence of SEQ ID NO;

26 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

or b) SEQ ID NO;

3 or its complement;

wherein the sequence of SEQ ID NO;

3 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides;

a nucleic acid probe which is capable of hybridising to the NGO1012 sequence or a fragment thereof;

a nucleic acid probe which is capable of hybridising to the NGO1642 sequence or a fragment thereof;

a nucleic acid probe comprising a nucleic acid sequence which comprises 20 or more contiguous nucleotides selected from SEQ ID NO;

8 or its complement;

wherein the sequence of SEQ ID NO;

8 or its complement may be mutated by up to 5 additions, deletions and/or substitutions of single nucleotides; and

/or

a composition comprising a forward primer as defined above and a reverse primer as defined above, optionally further comprising a nucleic acid probe as defined above, optionally further comprising a Pectobacterium atrosepticium nucleic acid sequence for use as an internal positive control, optionally wherein said Pectobacterium atrosepticium is of the strain ATCC BAA-672, optionally further comprising a forward control primer and reverse control primer which are capable of amplifying the Pectobacterium atrosepticium nucleic acid sequence, optionally further comprising a control nucleic acid probe which is capable of hybridising to the Pectobacterium atrosepticium nucleic acid sequence.

17. The forward primer, reverse primer, pair of primers or nucleic acid probe of claim 15, wherein:

the forward primer comprises the nucleic acid sequence of SEQ ID NO;

4;

the reverse primer comprises the nucleic acid sequence of SEQ ID NO;

27 or SEQ ID NO;

5;

the forward primer comprises the nucleic acid sequence of SEQ ID NO;

9;

the reverse primer comprises the nucleic acid sequence of SEQ ID NO;

10;

the nucleic acid probe comprises a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO;

26 or SEQ ID NO;

3;

the nucleic acid probe consists of the nucleic acid sequence of SEQ ID NO;

26 or SEQ ID NO;

3;

the nucleic acid probe comprises a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO;

8;

the nucleic acid probe consists of the nucleic acid sequence of SEQ ID NO;

8;

the primer(s) and/or the nucleic acid probe comprises a minor groove binding moiety;

or comprises at least one locked nucleic acid (LNA) nucleotide; and

/or

the primer(s) or nucleic acid probe is conjugated to an electrochemically active label.

16. (canceled)

18-34. -34. (canceled)

36. (canceled)

39-46. -46. (canceled)

47. A method selected from the group consisting of:

a method of detecting the presence of Neisseria gonorrhoeae in a sample comprising a step of amplifying a first target sequence and a second target sequence and a step of detecting the first target sequence and the second target sequence, wherein the first target sequence consists of SEQ ID NO;

2 and the second target sequence consists of SEQ ID NO;

7, and said step of detecting comprises hybridising the first target sequence to a nucleic acid probe comprising SEQ ID NO;

3 and a ferrocene label, hybridising the second target sequence to a nucleic acid probe comprising SEQ ID NO;

8 and a ferrocene label, and identifying the occurrence of hybridisation by detecting the state of the labelled probes, and wherein said step of amplifying the first target sequence and the second target sequence uses the polymerase chain reaction and involves the use of forward primers consisting of SEQ ID NOs;

4 and 10 and reverse primers consisting of SEQ ID NOs;

5 and 11;

a method of detecting the presence of Neisseria gonorrhoeae in a sample comprising a step of amplifying a first target sequence and a second target sequence and a step of detecting the first target sequence and the second target sequence, wherein the first target sequence consists of SEQ ID NO;

25 and the second target sequence consists of SEQ ID NO;

7, and said step of detecting comprises hybridising the first target sequence to a nucleic acid probe comprising SEQ ID NO;

26 and a ferrocene label, hybridising the second target sequence to a nucleic acid probe comprising SEQ ID NO;

8 and a ferrocene label, and identifying the occurrence of hybridisation by detecting the state of the labelled probes, and wherein said step of amplifying the first target sequence and the second target sequence uses the polymerase chain reaction and involves the use of forward primers consisting of SEQ ID NOs;

4 and 10 and reverse primers consisting of SEQ ID NOs;

27 and 11;

a method of diagnosis of gonorrhoea comprising a method or using a primer, probe or composition; and

/or

a method for detecting N. gonorrhoeae, C. trachomatis and T. vaginalis in a sample.

48-49. -49. (canceled)

51-53. -53. (canceled)

55-58. -58. (canceled)