Detection of human papillomavirus e6 mrna

• US 20050244813A1
• Filed: 01/07/2003
• Published: 11/03/2005
• Est. Priority Date: 01/07/2002
• Status: Abandoned Application

• Current Assignee: Frank Karlsen
• Original Assignee: Frank Karlsen
• Inventors: Karlsen, Frank
• Application Number:
  US10/500,831
• Publication Number:
  US 20050244813A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/708 for papilloma

Abstract

An oligonucleotide molecule for use in the detection of mRNA transcribed from the E6 gene of a human papillomavirus, the oligonucleotide comprising any one of sequence numbers 1-133.

Claims

1. An oligonucleotide molecule for use in the detection of mRNA transcribed from the E6 gene of a human papillomavirus, the oligonucleotide comprising any one of sequence numbers 1-133.

2. An oligonucleotide molecule according to claim 1 which is an oligonucleotide primer selected from:

(i) a NASBA P1 primer comprising one of sequence numbers 2, 4, 8, 11, 14, 17, 20, 22, 25, 28, 31, 34, 37, 39, 42, 45, 48, 50, 52, 57, 60, 63, 65, 69, 71, 75, 78, 81, 84, 87, 90, 92, 96, 98, 100, 105, 107, 111, 113, 115, 121, 126, 127, 128 or 129;

(ii) a NASBA P2 primer comprising one of sequence numbers 1, 3, 7, 10, 13, 16, 19, 21, 24, 27, 30, 33, 36, 38, 41, 44, 47, 49, 51, 56, 62, 64, 68, 70, 74, 77, 80, 83, 86, 89, 91, 95, 97, 99, 104, 106, 110, 112, 114, 120, 103, 131, 132 or 133; and

(iii) a PCR primer comprising one of sequence numbers 1, 3, 7, 10, 13, 16, 19, 21, 24, 27, 30, 33, 36, 38, 41, 44, 47, 49, 51, 56, 59, 62, 64, 68, 70, 74, 77, 80, 83, 86, 89, 91, 95, 97, 99, 104, 106, 110, 112, 114, 120, 2, 4, 8, 11, 14, 17, 20, 22, 25, 28, 31, 34, 37, 39, 42, 45, 48, 50, 52, 57, 60, 63, 65, 69, 71, 75, 78, 81, 84 87, 90, 92, 96, 98, 100, 105, 107, 111, 113, 115, 121, 126, 127, 128, 129, 130, 131, 132 or 133;

3. An oligonucleotide primer according to claim 2 which is a NASBA P1 primer having the sequence AATTCTAATACGACTCACTATAGGGAGAAGG-SEQ, wherein SEQ represents any one of sequence numbers 2, 4, 8, 11, 14, 17, 20, 22, 25, 28, 31, 34, 37, 39, 42, 45, 48, 50, 52, 57, 60, 63, 65, 69, 71, 75, 78, 81, 84 87, 90, 92, 96, 98, 100, 105, 107, 111, 113, 115, 121, 126, 127, 128 or 129, and wherein AATTCTAATACGACTCACTATAGGGAGAAGG is SEQ ID NO:

385.

4. An oligonucleotide primer according to claim 2 which is a NASBA P2 primer having the sequence GATGCAAGGTCGCATATGAG-SEQ wherein SEQ represents any one of sequence numbers 1, 3, 7, 10, 13, 16, 19, 21, 24, 27, 30, 33, 36, 38, 41, 44, 47, 49, 51, 56, 59, 62, 64, 68, 70, 74, 77, 80, 83, 86, 89, 91, 95, 97, 99, 104, 106, 110, 112, 114, 120, 130, 131, 132 or 133, and wherein GATGCAAGGTCGCATATGAG is SEQ ID NO:

387.

5. An oligonucleotide molecule according to claim 1 which is a probe for use in the detection of mRNA transcribed from the E6 gene of a human papillomavirus comprising one of sequence numbers:

5, 6, 9, 12, 15, 18, 23, 26, 29, 32, 35, 40, 43, 46, 53, 54, 55, 58, 61, 66, 67, 72, 73, 76, 82, 85, 88, 93, 94, 101, 102, 103, 108, 109, 116, 117, 118, 119, 122, 130, 131, 132 or 133.

30. An oligonucleotide molecule according to claim 5 which is a molecular beacon probe.

6-7. -7. (canceled)

8. An oligonucleotide primer-pair for use in the detection of mRNA transcripts from the E6 gene of HPV 31, comprising:

a NASBA P2 primer comprising sequence number 30 and a NASBA P1 primer comprising sequence number 31.

9. An oligonucleotide primer-pair for use in the detection of mRNA transcripts from the E6 gene of HPV 33, comprising:

a NASBA P2 primer comprising sequence number 38 and a NASBA P1 primer comprising sequence number 39.

10-15. -15. (canceled)

16. An oligonucleotide primer-pair for use in the detection of mRNA transcripts from the E6 gene of HPV 45, comprising:

a NASBA P2 primer comprising sequence number 89 and a NASBA P1 primer comprising sequence number 90.

21. A primer/probe set comprising a primer-pair according to any one of claims 8, 9 or 16 and at least one oligonucleotide probe specific for amplification products generated using the primer-pair.

22. A method of detecting HPV mRNA in a test sample suspected of containing HPV which comprises performing an a nucleic acid sequence based amplification (NASBA) reaction on a preparation of nucleic acid isolated from the test sample to amplify a portion of the mRNA transcribed from the E6 gene of HPV, wherein the amplification reaction is performed using a primer-pair according to any one of claims 8, 9 or 16.

25. A method according to claim 22 which comprises:

(a) assembling a reaction mixture comprising said primer-pair, an RNA directed DNA polymerase, a ribonuclease that hydrolyses the RNA strand of an RNA-DNA hybrid without hydrolysing single or double stranded RNA or DNA, an RNA polymerase that recognises said promoter, and ribonucleoside and deoxyribonucleoside triphosphates;

(b) incubating said reaction mixture with a preparation of nucleic acid isolated from a test sample suspected of containing HPV under reaction conditions which permit a NASBA amplification reaction; and

(c) detecting and/or quantitatively measuring any HPV-specific product of the NASBA amplification reaction.

26. A method according to claim 25 wherein step (c) comprises real-time detection of an HPV-specific product of the NASBA amplification reaction.

27. A method according to claim 25 wherein the reaction mixture further comprises a molecular beacons probe oligonucleotide and the formation of any HPV-specific NASBA product in the NASBA reaction is monitored by detecting fluorescence from the fluorescent moiety included in the molecular beacons probe.

29. A reagent kit for use in the detection of HPV by NASBA, the kit comprising an oligonucleotide primer-pair as defined in any one of claims 8, 9 or 16 and optionally an enzyme mixture comprising an RNA directed DNA polymerase, a ribonuclease that hydrolyses the RNA strand of an RNA-DNA hybrid without hydrolysing single or double stranded RNA or DNA, and an RNA polymerase that recognises the promoter sequence present in at least one NASBA P1 primer oligonucleotide included in the reagent kit.

31. A method according to claim 26 wherein the reaction mixture further comprises a molecular beacons probe oligonucleotide and the formation of any HPV-specific NASBA product in the NASBA reaction is monitored by detecting fluorescence from the fluorescent moiety included in the molecular beacons probe.

17-20. -20. (canceled)

23-24. -24. (canceled)

28. (canceled)