Detection of nucleic acids from multiple types of human papillomaviruses
View Patent ↗Nucleic acid oligonucleotide sequences are disclosed which include amplification oligomers and probe oligomers which are useful for detecting multiple types of human papillomaviruses (HPV) associated with cervical cancer. Methods for detecting multiple HPV types in biological specimens by amplifying HPV nucleic acid sequences in vitro and detecting the amplified products are disclosed.
1. A multiplex method of detecting the presence of type A1 and type C1 human papillomavirus (HPV) nucleic acids present in a biological sample, comprising the steps of:
(a) treating a biological sample suspected of containing HPV type A1 or HPV type C1 to release intracellular components;
(b) separating an HPV nucleic acid away from a component of the biological sample;
(c) contacting the separated HPV nucleic acid with a combination of amplification oligonucleotides that amplify a HPV type A1 target sequences and a combination of amplification oligomers that amplify HPV type C1 target sequences in an E6/E7 target region sequence, wherein the combination of amplification oligomers configured to amplify HPV type A1 comprises amplification oligomers consisting essentially of SEQ ID NOS:18, 19, 20, 21, 22, 23, 42 and 38
(d) amplifying a HPV target sequence by using the amplification oligonucleotides and a nucleic acid polymerase in vitro to produce an HPV amplified product; and
(e) detecting the amplified product to indicate the presence in the sample of at least one of HPV types A1 or C1.
2. The method of claim 1 , wherein the step of separating an HPV nucleic acid from components of the sample uses at least two capture oligonucleotides, wherein a capture oligonucleotide for separating HPV type A1 nucleic acid is SEQ ID NO:2 joined to a ligand moiety.
3. The method of claim 2 , wherein the ligand moiety joined to the capture oligonucleotide is a nucleic acid that is non-complementary to an HPV sequence.
4. The method of claim 1 , wherein the amplifying step uses an amplification process that is substantially isothermal.
5. The method of claim 1 , wherein the amplifying step uses a transcription-associated amplification method.
6. The method of claim 1 , wherein the detecting step uses a combination of probe oligonucleotides and at least one probe oligonucleotide bound to the HPV amplified product results in a signal that indicates the presence of at least one of HPV types 16, 18, 31, 33, 35, 39, 45, 52, 58, 59 and 68 in the biological sample.
7. The method of claim 6 , wherein the detecting step uses a combination of probe oligonucleotides configured to detect amplification product generated in step (d), wherein the combination of probe oligonucleotides includes SEQ ID NO:11, and wherein each probe oligonucleotide joined directly or indirectly to a label.
8. The method of claim 6 , wherein the detecting step uses a combination of probe oligonucleotides configured to detect amplification product generated in step (d), wherein the combination of probe oligonucleotides includes SEQ ID NO:44, SEQ ID NO:45 or SEQ ID NO:46 for detecting amplified product of HPV type C1.
9. The method of claim 1 , wherein the contacting step further includes introducing a non-HPV internal control sequence into the sample, wherein the amplifying step further includes amplifying the non-HPV internal control sequence to produce an amplified internal control sequence, and wherein the detecting step further includes detecting the amplified internal control sequence to produce a signal that indicates that the method steps have been performed appropriately.
10. The method of claim 3 , wherein the capture oligonucleotide is SEQ ID NO:1.
11. The method of claim 1 , wherein the combination of amplification oligomers configured to amplify HPV type C1 includes a first amplification oligomer that is SEQ ID NO:29 or SEQ ID NO:31 and a second amplification oligomer that is SEQ ID NO:39.
12. The method of claim 11 , wherein the amplification oligomer that is SEQ ID NO:29 is joined at its 5′ end to a promoter sequence and wherein the amplification oligomer that is SEQ ID NO:31 is joined at its 5′ end to a promoter sequence.
13. The method of claim 1 , wherein the combination of amplification oligomers configured to amplify HPV type C1 is SEQ ID NOS:28 and 39; SEQ ID NOS:30 and 39 or SEQ ID NOS:28, 30 and 39.
14. The method of claim 1 , wherein the biological sample is treated to physically disrupt the sample to release the intracellular components.
15. The method of claim 1 , wherein the biological sample is treated with an enzyme to release intracellular components.
16. The method of claim 1 , wherein the detection reaction is an end-point detection reaction.
17. An amplification reaction mixture for use in a multiplex method of amplifying type A1 and type C1 human papillomavirus (HPV) nucleic acids present in a biological sample, wherein the reaction mixture contains (i) amplification oligomers configured to generate an amplification product from HPV type Al that consist essentially of SEQ ID NOS:18, 19, 20, 21, 22, 23, 38 and 42 and (ii) amplification oligomers for generating an amplification product from HPV type C1.
18. The reaction mixture of claim 17 , wherein the amplification oligomers configured to generate an amplification product from HPV type C1 generate a type C1 amplification product that can be detected by one or more of detection probes SEQ ID NOS:44, 45 or 46.
19. The amplification reaction mixture of claim 18 , wherein the type C1 amplification product was generated with an amplification oligomer consisting essentially of SEQ ID NO:39.
20. The amplification reaction mixture of claim 18 , wherein the type C1 amplification product was generated with an amplification oligomer consisting essentially of SEQ ID NO:28.
21. The amplification reaction mixture of claim 20 , wherein SEQ ID NO:28 is joined at its 5 ′ end to a promoter sequence.
22. A method of detecting the presence of type A1 and type C1 human papillomavirus (HPV) nucleic acids present in a biological sample, comprising the steps of:
(a) treating a biological sample suspected of containing HPV type A1 or HPV type C1 to release an HPV nucleic acid;
(b) separating the HPV nucleic acid away from a component of the biological sample;
(c) contacting the separated HPV nucleic acid with:
(i) a combination of amplification oligonucleotides that amplify an HPV type A1 target sequence, the amplification oligonucleotides consisting essentially of SEQ ID NOS:18, 19, 20, 21, 22, 23, 42 and 38; and
(ii) a combination of amplification oligonucleotides that amplify an HPV type C1 target sequence, the amplification oligonucleotides consisting essentially of SEQ ID NOS:28 or 30 and SEQ ID NO:39;
(d) amplifying a HPV target sequence by using the amplification oligonucleotides from step (c) and a nucleic acid polymerase to produce an HPV amplification product; and
(e) detecting the amplification product to indicate the presence in the sample of at least one of HPV types A1 or C1.
23. The method of claim 22 , wherein the treating step physically disrupts the sample to release the nucleic acid.
24. The method of claim 22 , wherein the separating step uses one or more capture oligonucleotides.
25. The method of claim 22 , wherein the amplification step is a transcription-associated amplification.
26. The method of claim 22 , wherein the amplification step amplifies the HPV type A1 and the HPA type C1 nucleic acids in the same reaction well.
27. The method of claim 22 , wherein the detection step is an end-point detection reaction.
28. The method of claim 27 , wherein the detection reaction uses probe oligonucleotides.
29. The method of claim 27 , wherein the detection reaction uses two or more probe oligonucleotides selected from the group consisting of SEQ ID NOS:11, 44, 45 and 46.