Compositions, reaction mixtures and methods for detecting nucleic acids from type A1 and/or type C1 human papillomavirus
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 comprising nucleic acid sequences consisting essentially of SEQ ID NOS:18, 20, 22, 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 sequence for separating HPV type A1 nucleic acid is SEQ ID NO:2 joined to a ligand moiety or SEQ ID NO:1.
3. The method of claim 1 , wherein the amplifying step uses an amplification process that is substantially isothermal.
4. The method of claim 1 , wherein the detecting step uses a combination of probe oligonucleotides configured to detect amplification product generated in step (d) and thereby indicate the presence of at least one of HPV types 16, 18, 31, 35, 45, and 59 in the biological sample, and wherein the combination of probe oligomers includes one or more probes having a sequence of SEQ ID NO:11, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46.
5. The method of claim 4 , wherein each probe oligonucleotide is joined directly or indirectly to a label.
6. The method of claim 1 , wherein the combination of amplification oligomers configured to amplify HPV type C1 comprises a first amplification oligomer nucleic acid sequences that is SEQ ID NO:29, joined at its 5′ end to a promoter sequence, or SEQ ID NO:31, joined at its 5′ end to a promoter sequence, and a second amplification oligomer that is SEQ ID NO:39.
7. The method of claim 1 , wherein the combination of amplification oligomers configured to amplify HPV type C1 comprise sequences that are SEQ ID NOS:28 and 39; SEQ ID NOS:30 and 39 or SEQ ID NOS:28, 30 and 39.
8. The method of claim 1 , wherein the biological sample is treated to physically disrupt the sample to release the intracellular components.
9. The method of claim 1 , wherein the biological sample is treated with an enzyme to release intracellular components.
10. 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 A1, wherein the amplification oligomers comprise sequences that that consist essentially of SEQ ID NOS:18, 20, 22, and 38 and (ii) amplification oligomers for generating an amplification product from HPV type C1.
11. The reaction mixture of claim 10 , 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.
12. 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 comprising sequences consisting of SEQ ID NOS:18, 20, 22, and 38; and
(ii) a combination of amplification oligonucleotides that amplify an HPV type C1 target sequence, the amplification oligonucleotides comprising sequences 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.
13. The method of claim 12 , wherein the treating step physically disrupts the sample to release the nucleic acid.
14. The method of claim 12 , wherein the separating step uses one or more capture oligonucleotides.
15. The method of claim 12 , wherein the amplification step is a transcription-associated amplification.
16. The method of claim 12 , wherein the amplification step amplifies the HPV type A1 and the HPV type C1 nucleic acids in the same reaction well.
17. The method of claim 12 , wherein the detection reaction uses probe oligonucleotides.
18. The method of claim 12 , wherein the detection reaction uses two or more probe oligonucleotides comprising sequences selected from the group consisting of SEQ ID NOS:11, 44, 45 and 46.