Compositions, reaction mixtures and methods for detecting nucleic acids from multiple types of 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 method of detecting multiple types of human papillomavirus (HPV) nucleic acid present in a biological sample, comprising the steps of:
(a) contacting nucleic acid in a biological sample with a mixture of amplification oligomers, each of which amplifies a HPV sequence in an E6/E7 target region sequence, in which the mixture comprises:
(i) first amplification oligomers comprising nucleic acid sequences selected from the group consisting of SEQ ID Nos. 18, 20, 22, 24, 28, 30, 32, 34, 36, RNA equivalents thereof, complements thereof, and combinations thereof; and
(ii) second amplification oligomers comprising nucleic acid sequences selected from the group consisting of SEQ ID Nos. 38, 39, 40, 41, RNA equivalents thereof, complements thereof, and combinations thereof;
(b) amplifying a HPV sequence from the target region sequence in at least one HPV type by using the amplification oligomers and a nucleic acid polymerase in vitro to produce an HPV amplified product; and
(c) detecting the amplified product by using a detection probe oligomer that is sufficiently complementary to hybridize specifically with the HPV amplified product to indicate the presence in the sample of at least one type of HPV nucleic acid.
2. The method of claim 1 , further comprising a step of separating RNA of at least one of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 from other components in the sample by contacting HPV RNA in the sample with a capture oligomer and separating a complex that includes the capture oligomer and HPV RNA from other components in the sample before the amplifying step.
3. The method of claim 2 , wherein the capture oligomer is present in a mixture of capture oligomers made up of at least two capture oligomers in which individual oligomer sequences are selected from the group consisting of SEQ ID Nos. 1 to 10, which includes the complementary oligomer sequences or RNA equivalents of the specified sequences, and wherein each of SEQ ID Nos. 2, 4, 6, 8 10, RNA equivalents thereof and complements thereof further comprise a ligand moiety joined to the oligomer 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 detecting step uses a mixture of probe oligomers in which at least one probe oligomer binds specifically to the HPV amplified product and results in a signal to indicate the presence in the sample of at least one of the HPV types.
6. The method of claim 5 , wherein the detecting step uses a mixture of probe oligomers comprising at least two detection probe oligomers comprising sequences selected from the group consisting of SEQ ID Nos. 11 to 17, complementary oligomer sequences, RNA equivalents of the oligomer sequences, and combinations thereof.
7. The method of claim 1 , wherein the first amplification oligomers are at least three amplification oligomers comprising nucleic acid sequences selected from the group consisting of SEQ ID Nos. 18, 20, 22, 24, 28, 30, 32, 34, 36, RNA equivalents thereof, complements thereof, and combinations thereof and the second amplification oligomers are at least three amplification oligomers comprising nucleic acid sequences selected from the group consisting of SEQ ID Nos. 38, 39, 40, 41, RNA equivalents thereof, complements thereof, and combinations thereof.
8. The method of claim 7 , wherein the second amplification oligomers are at least four amplification oligomers comprising the nucleic acid sequence, the RNA equivalent sequence, and/or the complement of SEQ ID Nos. 38, 39, 40 and 41.