Detection of nucleic acids from multiple types of human papillomavirus

Abstract

To provide detection of nucleic acids from multiple types of human papillomavirus.SOLUTION: The present invention includes compositions and methods for detection of nucleic acid sequences in multiple HPV types that may be present in a biological sample. Nucleic acid sequences and methods for detecting plural types of human papillomavirus (HPV) nucleic acid sequences contained in biological samples are disclosed. Nucleic acid oligomers for amplifying HPV nucleic acids of the multiple types in vitro, and a kit accommodating nucleic acid detection probe sequences for detecting the amplified HPV sequences are also disclosed.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority under 35 U.S.C. 119(e) to Provisional Patent Application No. 60 / 634,458, filed December 8, 2004, which is incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present invention relates to the diagnostic detection of infectious agents associated with cancer risk, in particular compositions and assay methods for detecting human papillomavirus (HPV) by in vitro nucleic acid amplification and detection assay methods for detecting nucleic acid sequences. [Background technology]

[0003] Human papillomaviruses (HPV) target epithelial tissues for infection and are responsible for a variety of cancers, primarily squamous cell carcinoma and adenocarcinoma. HPV-associated cancers include those of the head and neck (larynx, oral cavity, oropharynx, tonsils and esophagus), respiratory tract, breast, skin, cervix and anus. Although HPV infection appears to be a necessary factor for the development of certain cancers, other factors may also influence carcinogenesis (Braakhuise et al., 2004, J. Natl. Cancer Inst. 96(13): 998-1006; Dahlstrandet al., 2004, Anticancer Res. 24(3b): 1829-35; Daling et al., 2004, Cancer 101(2): 270-80; Ha et al., 2004, Crit. Rev. Oral Biol. Med. 15(4): 188-96; Hafkamp et al., 2004, Crit. Rev. Oral Biol. Med. 15(4): 188-96). etal.,Acta Otolaryngol. 124(4): 520-6; Harwoodetal.,2004,Br.J.Dermatol.150(5):949-57; Rees et al., 2004,Clin.Otolaryngol.29(4):301-6;Widschwendter et al. al., 2004, J. Clin. Virol. 31(4): 292-7).

[0004] At least 77 different types of HPV have been identified. Of these, HPV16 and HPV18 are frequently implicated in various HPV-associated cancers, although the risk levels associated with HPV types may vary according to different morphologies of papilloma-associated cancers. To elucidate the relationship of HPV infection to cancer, the pathogenesis of human papillomaviruses in epithelia has been studied. HPV infects basal cells of stratified epithelia and establishes as multicopy episomes or integrated genomes, whereby viral DNA is replicated along with cellular chromosomes (reviewed by Longworth et al., 2004, Microbiol. MoI. Biol. Rev. 68(2):362-72). Cellular division During cleavage, daughter cells migrate from the basal layer and differentiate, during which vegetative HPV viral replication and late gene expression are activated to produce progeny HPV. Although the immune system of an infected individual usually clears the HPV infection within 1-2 years, infected basal cells may persist for decades. HPV infection leads to chromosomal instability and aneuploidy, which promotes HPV integration (Melsheimer et al., 2004, Clin. Cancer Res. 10(9): 3059-63; Reidy et al., 2004, Laryngoscope114(11):1906-9). During HPV genome integration, HPVE2 Gene disruption can result in deregulated expression of the HPVE6 / E75 oncogene, which encodes an oncoprotein that targets the regulatory proteins pRb and p53. Thus, a series of events that alter cellular regulation can result in carcinogenesis (Braunet al., 2004, Cancer Lett. 209(1): 37-49; Fan et al., 2004, Crit. Rev. Eukaryot. Gene Expr. 14(3): 183-202; Fiedler et al., 2004, FASEB J. 18(10): 1120-2; Psyrri et al., 2004, Cancer Res., 64(9): 3079-86; Si et al., 2004, J. Clin. Virol. 32(1): 19-23). .

[0005] The incidence of cervical cancer is high worldwide, with an estimated 450,000 new cases per year, and the association between HPV infection and cervical cancer has been the subject of many studies and epidemiological investigations. High-risk HPV types (HR-HPV) associated with cervical cancer include HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 73, although the epidemiological importance of individual types may vary in different geographical regions or depending on clinical trial parameters (Munozet al.,2004,Int. J. Cancer 111(2) : 278-85; Chaturvedi et al.,2005,J.Med.Virol.75(1):105-13; Smith et al., 2004,Int. J. Gynaecol. Obstet. 87(2):131-7). In general, the risk of developing cervical cancer is low (LR-HP V) Possible HPV infections include HPV types 6, 11, 43, 43, 44, 61, 71, and 72.

[0006] In HPV-infected women, cervical infection can lead to condylomata (genital warts), cervical intraepithelial neoplasia (CIN), and cervical cancer (Kahnetal., 2004, Adolesc. Med. Clin 15(2):301-21, ix). Cytological examination of cervical cells is the standard method for cervical cancer screening in many countries. HPV is the main screening tool for the detection of cancer, and the CIN grading system (1-3) is usually used to monitor precancerous lesions for treatment decisions and / or subsequent monitoring. In addition to cytological screening, molecular screening for HPV nucleic acid can be a cost-effective prognostic test that can extend the interval between cytological examinations (Wiley et al., 2004, Curr. Oncol. Rep. 6(6): 497-506; Zielinski et al., 2004, Obstet. Gynecol. Surv. 59(7):543-53; Clavel et al., 2004, Br. J. Cancer 90(9):1803-8). Molecular assay methods have been developed for the detection of cancer in humans. HPV immunoassays have been developed to detect specific HPV proteins and nucleic acid sequences in biological specimens, such as Papanicolaou smears (Chen et al., 2005, Gynecol. Oncol. 99(3): 578-84; Carozzi et al. al., 2005, Am. J. Clin. Pathol. 124(5):716-21;Molden et al., 2005, Cancer Epidemiol. Biomarkers Prev.14(2): 367-72;Asato et al., 2004,J.Infect.Dis.189(1):1829-32;Federschneider et al., 2004,Am. J.Obstet.Gynecol.191(3):757-61;Remmerbach et al., J. Clin. Virol. 30(4):302-8).

[0007] Vaccination against common HPV types may be useful in treating or preventing genital warts, or in preventing the development of cancer, particularly cervical cancer. Various forms of HPV vaccination are in development or testing (Ault et al., 2004, Vaccine 22(23-24): 3004-7; Corona Gutierrez et al., 2004, Hum.Genet.Ther. 15(5): 421-31; Harper et al.,2004,Lancet 364(9447): 1757-65; Roden et al., 2004, Hum. Pathol. 35(8):971-82). Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need to effectively and sensitively detect the presence of HPV in biological specimens to provide physicians with diagnostic and prognostic information for HPV-infected patients, particularly women infected with HR-HPV types in cervical tissue. There is also a need to effectively and sensitively detect the presence of HPV in biological specimens obtained from vaccinated individuals for HPV infection to determine the short-term and long-term efficacy of vaccination. [Means for solving the problem]

[0009] Summary of the Invention The present invention includes compositions and methods for detecting nucleic acid sequences of multiple HPV types that may be present in a biological sample.

[0010] In one aspect of the invention, an amplification oligomer mixture is provided, wherein each oligomer sequence in the mixture is selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 42, including complementary oligomer sequences or RNA equivalents of said specific sequences. Preferred embodiments of the mixture include first amplification oligomers of SEQ ID NO: 19 or 42, 21, 23, 25, 27, 29, 31, 33 and 35, and second amplification oligomers of SEQ ID NO: 37, 38, 39, 40 and 41, or complementary oligomer sequences or RNA equivalents of said oligomer sequences. Other preferred embodiments include first amplification oligomers of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36, and second amplification oligomers of SEQ ID NO: 37, 38, 39, 40 and 41, or complementary oligomer sequences or RNA equivalents of said oligomer sequences. Other preferred embodiments include first amplification oligomers of SEQ ID NOs: 19 or 42, 21, 23, 25, 27, 29, 31, 33, and 35, and second amplification oligomers of SEQ ID NOs: 38, 39, 40, and 41, or complementary oligomer sequences or RNA equivalents of said oligomer sequences. Other preferred embodiments include first amplification oligomers of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, and second amplification oligomers of SEQ ID NOs: 38, 39, 40, and 41, or complementary oligomer sequences or RNA equivalents of said oligomer sequences. In some embodiments of the oligomer mixture, one or more individual oligomers include a backbone that includes at least one 2'-methoxy RNA group, at least one 2'-fluoro substituted RNA group, at least one peptide nucleic acid linkage, at least one phosphorothioate linkage, at least one methylphosphonate linkage, or any combination of the foregoing. The mixture embodiment may be contained in a kit.

[0011] In another aspect of the invention, there is provided an oligomer mixture, wherein each oligomer sequence in the mixture is selected from the group consisting of SEQ ID NO: 11 to SEQ ID NO: 17, SEQ ID NO: 44 to SEQ ID NO: 54, and SEQ ID NO: 58, the group including complementary oligomer sequences or RNA equivalents of the specific sequences. A preferred embodiment of the mixture includes SEQ ID NO: 11 to 17, or complementary oligomer sequences or RNA equivalents of the oligomer sequences. Other preferred embodiments include SEQ ID NO: 11 to 15 and 17, or complementary oligomer sequences or RNA equivalents of the oligomer sequences. Other preferred embodiments include oligomers of SEQ ID NO: 11 to 15 and 17, and at least one of SEQ ID NO: 44 to SEQ ID NO: 54 and SEQ ID NO: 58, or complementary oligomer sequences or RNA equivalents of the oligomer sequences. Other preferred embodiments include SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45 and 52, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences. Preferred embodiments of the mixture include those in which each oligomeric sequence comprises a label attached directly or indirectly to said oligomer. In a preferred embodiment, each oligomeric sequence comprises a label that is a chemiluminescent compound, more preferably an acridinium ester (AE) compound. In a preferred embodiment of the mixture, each oligomeric sequence has a backbone that includes at least one 2'-methoxy RNA group. The above embodiments of the mixture may be contained in a kit.

[0012] In another aspect of the invention, a mixture of at least two oligomers is provided, wherein each oligomer sequence in the mixture is selected from the group consisting of SEQ ID NOs: 1-10, including complementary oligomer sequences or RNA equivalents of the specific sequences. A preferred embodiment of the mixture includes at least two oligomers selected from SEQ ID NOs: 2, 4, 6, 8, and 10, with a ligand moiety attached to each oligomer. Another preferred embodiment of the mixture includes oligomers of SEQ ID NOs: 2, 4, 6, 8, and 10, with a ligand moiety attached to each oligomer. Another preferred embodiment of the mixture includes at least two oligomers selected from SEQ ID NOs: 1, 3, 5, 7, and 9. Another preferred embodiment of the mixture includes oligomers of SEQ ID NOs: 1, 3, 5, 7, and 9. In a preferred embodiment, at least one oligomer has a backbone that includes at least one 2'-methoxy RNA group, at least one 2'-fluoro substituted RNA group, at least one peptide nucleic acid linkage, at least one phosphorothioate linkage, or at least one methylphosphonate linkage.Preferred embodiments of the mixture are contained in a kit.

[0013] In another aspect of the invention, there is provided a method for detecting human papillomavirus (HPV) nucleic acid present in a biological sample, comprising the steps of: contacting nucleic acid in the biological sample containing RNA of at least one of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 with a mixture of amplification oligomers that amplify HPV sequences within the E6 / E7 target region sequence, wherein the mixture contains a first amplification oligomer and a second amplification oligomer for amplifying the polymorphic HPV target region sequence. the amplification oligomers are comprised of: an HPV sequence derived from a target region sequence in at least one HPV type is amplified in vitro with the amplification oligomers and a nucleic acid polymerase to generate an HPV amplification product; and the amplification product is detected with a detection probe oligomer that is sufficiently complementary to specifically hybridize with the HPV amplification product to indicate the presence in the sample of at least one of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. In a preferred embodiment, the amplification step employs a first amplification oligomer of SEQ ID NO: 19 or 42, 21, 23, 25, 27, 29, 31, 33, and 35, and a second amplification oligomer of SEQ ID NO: 37, 38, 39, 40, and 41, or a complementary oligomer sequence or RNA equivalent of said oligomer sequences. In another preferred embodiment, the amplification step uses first amplification oligomers of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36 and second amplification oligomers of SEQ ID NOs: 37, 38, 39, 40 and 41 or complementary oligomer sequences or RNA equivalents of said oligomer sequences. In a preferred embodiment, the amplification step uses first amplification oligomers of SEQ ID NOs: 19 or 42, 21, 23, 25, 27, 29, 31, 33 and 35 and second amplification oligomers of SEQ ID NOs: 38, 39, 40 and 41 or complementary oligomer sequences or RNA equivalents of said oligomer sequences. In another preferred embodiment, the amplification step uses first amplification oligomers of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36 and second amplification oligomers of SEQ ID NOs: 38, 39, 40 and 41 or complementary oligomer sequences or RNA equivalents of said oligomer sequences.A preferred embodiment of this method further comprises, prior to the amplification step, a step of contacting HPV RNA in the sample with a capture oligomer to separate 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, and separating the complex containing the capture oligomer and HPV RNA from other components in the sample. In a preferred embodiment including this separation step, the capture oligomer is present in a capture oligomer mixture composed of at least two oligomers, and each oligomer sequence in the mixture is selected from the group consisting of SEQ ID NOs: 1 to 10, which include complementary oligomer sequences or RNA equivalents of the specific sequences. In a preferred embodiment of the method comprising this separation step, the capture oligomers are present in a capture oligomer mixture consisting of at least two oligomers selected from SEQ ID NOs: 2, 4, 6, 8, and 10, with a ligand moiety attached to each oligomer; or oligomers of SEQ ID NOs: 2, 4, 6, 8, and 10, with a ligand moiety attached to each oligomer; or at least two oligomers selected from SEQ ID NOs: 1, 3, 5, 7, and 9; or oligomers of SEQ ID NOs: 1, 3, 5, 7, and 9. In a preferred embodiment of this method, the amplification step employs an amplification method that is substantially isothermal. In a preferred embodiment of this method, the amplification step employs a transcription-associated amplification method. In one embodiment of this method, the detection step employs a probe oligomer mixture, and at least one probe oligomer in the mixture specifically binds to an HPV amplification product to generate a signal indicative of the presence of at least one HPV type in the sample. In a preferred embodiment, the detection step uses a probe oligomer mixture consisting of SEQ ID NOs: 11 to 17, or complementary oligomer sequences of said oligomer sequences, or RNA equivalents. In another preferred embodiment of the detection step, the detection step uses a probe oligomer mixture consisting of SEQ ID NOs: 11 to 15 and 17, or complementary oligomer sequences of said oligomer sequences, or RNA equivalents.Another preferred embodiment of the detection step uses a probe oligomer mixture consisting of oligomers of SEQ ID NOs: 11-15 and 17, and at least one of SEQ ID NOs: 44-54 and SEQ ID NO: 58, or complementary oligomer sequences or RNA equivalents of said oligomer sequences. Another preferred embodiment of the detection step uses a probe oligomer mixture consisting of SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45 and 52, or complementary oligomer sequences or RNA equivalents of said oligomer sequences. One embodiment of the method further amplifies and detects a non-HPV-internal control sequence. A preferred embodiment including an internal control further includes the contacting step further introducing a non-HPV-internal control sequence to the sample, the amplifying step further amplifying the non-HPV-internal control sequence to generate an amplified internal control sequence, and the detecting step further includes detecting the amplified internal control sequence to generate a signal indicating that the step was performed properly.

[0014] The following description and the accompanying drawings, which form a part hereof, are intended to explain the principles of the invention. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is an evolutionary tree showing the genetic relationships of certain HPV types. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description of the Invention The present invention includes oligomer sequences and methods for detecting nucleic acid sequences from multiple HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) present in biological samples from humans. The sequences and methods are useful for diagnosing HPV infection, including persistent infection where detection of HPV sequences provides prognostic information related to carcinogenesis. The sequences and assay methods are also useful for monitoring the status of patients at risk for cancer and / or for monitoring the response of patients to treatment. Because the assay methods sensitively detect the presence of HPV nucleic acid, they are also useful for detecting vascular space invasion or lymph node metastasis of HPV-associated cancers, thus providing further prognostic information. The sequences and assay methods are also useful for monitoring the efficacy of anti-HPV vaccinations following vaccination of HPV-exposed individuals.

[0017] The targeted HPV types cumulatively represent high-risk types associated with cervical cancer. Preferably, the oligomer sequences are designed to detect more than one HPV type in the group by targeting common or highly similar E6 / E7 gene sequences among different HPV types, while avoiding undesirable oligonucleotide-oligonucleotide interactions that may occur when present in a mixture, for example, in an in vitro nucleic acid amplification reaction or hybridization detection reaction. The sequences are also designed not to detect related sequences found in HPV types identified as having low risk of cervical cancer (e.g., HPV types 6, 11, 42, 43, and 44). For comparison of the tagged sequences, the various HPV types were classified based on their genetic relationships as shown in FIG. 1 and are referred to herein as group A1, which includes HPV types 16, 31, and 35; group A2, which includes HPV types 33, 52, and 58; group B, which includes HPV types 6, 11, 42, 43, and 44; group C1, which includes HPV types 18, 45, and 59; and group D, which includes HPV types 51, 56, and 66. For sequence comparison, known sequences available from a public database (GenBank) were aligned manually or by computerized algorithms, and the aligned sequences were used to select oligonucleotide sequences for synthesis and testing that suggested potential oligomers that fit various criteria. These criteria included sequences in the range of about 15-30 nt in length, GC content, and predicted T of hybridization complexes containing the selected sequences. m, and the substantial absence of predictable secondary structures due to self-hybridization or intermolecular hybridization with other selected oligomers. The selected sequences were designed to specifically recognize the targeted HPV genomic or RNA sequences of one or preferably more than one HPV type in the group under substantially identical hybridization conditions for all selected oligonucleotide sequences. The known sequences used for alignment and selection included GenBank Accession Nos. AF092932, AF125673, AF404678, D90400, J04353, K02718, M12732, M27022, M73236, M14119, M62849, M74117, X05015, X74477, X74479, X74481, X74483, X77858, and Y13218. From the selected sequence, oligomers are synthesized in vitro by standard methods, e.g., by forming hybridization complexes containing the oligomer and a synthetic RNA that usually contains the complement of the oligomer sequence. m were experimentally determined to identify the synthetic oligomers and to characterize their functional properties in nucleic acid amplification and detection reactions using previously reported conditions (e.g., U.S. Patent Nos. 5,399,491 and 5,554,516, Kacian et al.; and 5,283,174, Arnold et al.). For use in the methods, the HPV-specific sequences can be linked to other sequences, such as 5' promoter sequences as shown in SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or 3' tail sequences as shown in SEQ ID NOs: 1, 3, 5, 7, and 9.

[0018] The following methodology was used to efficiently select oligomers that would function in an assay to detect multiple different HPV types. Oligomers that act as detection probes for HPV groups were first selected to determine the target regions of the HPV genome or RNA sequence to be amplified. Amplification oligomers were then selected for each candidate target region, synthesized, and tested in amplification reactions with individual HPV types of the group to be detected (e.g., HPV16, 31, and 35 of group A1). If the amplification and detection reactions yielded substantially similar positive results for each HPV type of a group, oligomers from different groups (e.g., A1 and A2) were combined in a multiplex amplification and detection reaction and each HPV type of the combined group was tested to determine whether the oligomer mixture functioned properly to yield positive results for each HPV type of the combined group, or whether adverse interactions (e.g., between oligomers in the mixture) reduced the sensitivity or specificity of the assay. If the assay results in the multiplex format were not acceptable, one or more oligomers were redesigned, synthesized, and tested in substantially the same manner. This process was repeated with various combinations of oligomers to obtain combinations of oligomers that function in a multiplex assay to amplify and detect HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. The multiplex assay method was further optimized using empirically determined optimal amounts of oligomers in the mixture to obtain positive results for each targeted HPV sequence while minimizing disruptive interactions between oligomers.

[0019] Selected oligomer sequences used to amplify and detect sequences of multiple HPV types are summarized in Table 1. For convenience, the sequences are given in HPV group designation, however, it will be understood that an oligomer sequence may also function to recognize other group type HPV sequences in a multiplex reaction. Although the sequences in Table 1 are shown as DNA sequences, it will be understood that the embodiments include sequences consisting of their corresponding RNA sequences, and sequences that are fully complementary to the disclosed DNA sequences or their corresponding RNA sequences.

[0020] [Table 1]

[0021] [Table 2]

[0022] [Table 3]

[0023] As described below, some of the oligomers are preferably used as amplification oligomers, including promoter primer aspects, some are preferably used as detection probes, and some are preferably used as capture probe oligomers to facilitate separation of HPV nucleic acid from other components of a biological sample prior to amplifying HPV target sequences. The capture probe oligomers preferably target sequences in the E6 / E7 gene sequence. In contrast, the amplification and detection oligomers target the E7 gene sequence. The oligomer sequences and methods using them are useful for detecting a number of different HPV types that are highly associated with cancer risk in humans. They are useful for detecting expression of the HPV genes E6 and E7, which, when constitutively expressed in human cells, result in cell cycle deregulation and cell proliferation that may progress to cancer. Because HPV E6 / E7 gene expression is suppressed in cells carrying episomal HPV, the assay methods and oligomer components of the present invention are designed to detect E6 / E7 RNA present in biological samples to provide prognostic information by detecting HPV expression associated with neoplasia and carcinogenesis.

[0024] In order that the present invention and its preferred embodiments may be understood, the following definitions are provided. Other scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. General definitions of terms can be found, for example, in the Dictionary of Microbiology and Molecular Biology, Vol. 2, pp. 111-115, 2001. (Singleton et al., 1994, John Wiley & Sons, New York, NY), or The HarperCollins Dictionary of Biology (Hale &Marham, 1991, Harper Perennial, New York, NY). Unless otherwise noted, any references herein to the same or similar term are incorporated herein by reference in their entirety. The techniques used are standard methods well known to those skilled in the art.

[0025] A "biological sample" is any tissue or material obtained from a living or dead human that may contain labeled nucleic acid. This includes, for example, laryngeal, oral, oropharyngeal, tonsillar, or esophageal tissue, respiratory or exudate samples; cervical or anal swab samples; biopsy tissues, including lymph node, gastrointestinal, stool, urine, semen, sputum, peripheral blood, plasma, serum, or other bodily fluids; tissue or material samples. Biological samples may be treated by well-known methods to physically or mechanically disrupt tissue or cellular structures and to release intracellular components into solutions that may contain enzymes, buffers, salts, detergents, and the like.

[0026] "Nucleic acid" refers to a polymeric compound (oligomer or polymer) containing nucleosides or nucleoside analogs containing nitrogenous bases or base analogs and linked together by phosphodiester bonds or other known bonds to form a polynucleotide. Nucleic acids include conventional ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or chimeric DNA-RNA and analogs thereof. The "backbone" of a nucleic acid may be composed of a variety of linkages, including one or more sugar-phosphodiester linkages, peptide-nucleic acid linkages (as in "peptide nucleic acid" or PNA; see WO 95 / 32305, Hydig-Hielsen et al.), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar portion of a nucleic acid may be ribose or deoxyribose or similar compounds with known substituents, such as 2'-methoxy and 2'-halide substituents (e.g., 2'-F). The nitrogenous bases are the following: the common bases (A, G, C, T, U), their analogs (e.g., inosine; The Biochemistry of the Nucleic Acids 5-36, Adams et al., 11th ed., 1992), proline, Derivatives of phosphorus or pyrimidine bases (e.g. N 4 -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituents at the 5- or 6-position, purine bases with altered or replaced substituents at the 2-, 6- and / or 8-positions, e.g., 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and O 4-alkyl-pyrimidines, and pyrazolo compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines; U.S. Pat. Nos. 5,378,825, 6,949,367, and WO 93 / 13121). Nucleic acids may contain "non-basic" residues that do not contain a nitrogenous base in the backbone for one or more residues (U.S. Pat. No. 5,585,481, Arnold et al.). Nucleic acids may contain only common sugars, bases, and linkages found in RNA and DNA, or may contain common components and substitutions (e.g., nucleic acids containing common bases attached by a 2'-methoxy backbone or a mixture of common bases and one or more base analogs). Nucleic acids may also include "locked nucleic acids" (LNAs), analogs containing one or more LNA nucleotide monomers in which the bicyclic furanose unit is locked into an RNA-mimetic sugar conformation, which enhances hybridization affinity to complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) (Vester et al., 2004, Biochemistry 43(42): 13233-41). Synthetic methods for producing the acids are well known in the art.

[0027] The interchangeable terms "oligomer" and "oligonucleotide" generally refer to nucleic acids having fewer than 1,000 residues, including a size range with a lower limit of about 2 to 5 nucleotides. Preferred oligomers fall within a size range with a lower limit of about 5 to about 15 nucleotides and an upper limit of about 60 to about 150 nucleotides. More preferably, the oligomers are in the size range of about 15 to about 100 nucleotides. Oligomers can be purified from natural sources, but are preferably synthesized by any known enzymatic or chemical method.

[0028] "Amplification oligonucleotide" or "amplification oligomer" refers to an oligomer that hybridizes to a target nucleic acid or the complementary sequence and participates in an in vitro nucleic acid amplification reaction. An amplification oligomer may be a "primer" or a "promoter-primer". A primer generally hybridizes to a template nucleic acid strand and has a 3' end that is extended in a polymerization reaction to generate a strand complementary to the template nucleic acid. The 5' region of the primer may be non-complementary to the target nucleic acid, e.g., it may contain a 5' promoter sequence, and the oligomer is referred to as a promoter-primer. Those skilled in the art will recognize that any oligomer that has a target complementary sequence and functions as a primer can be modified to include a 5' promoter sequence and function as a promoter-primer. Similarly, a promoter-primer can function as a primer independent of its promoter sequence. A preferred embodiment of an amplification oligonucleotide contains at least 10 contiguous bases, more preferably at least 12 contiguous bases, that are complementary to the target sequence (or the complementary strand). The contiguous bases are at least about 80%, more preferably at least about 90%, and most preferably about 100% complementary to the target region to which the oligomer hybridizes. The length of the amplification oligomer is preferably from about 10 to about 65 nucleotides and may contain modified nucleotides or base analogs.

[0029] "Amplification" refers to an in vitro method for obtaining multiple copies of a target sequence, its complement, or a fragment of a target sequence. Amplification of a "fragment" refers to the production of an amplified nucleic acid that contains less than the entire target region sequence or its complement. For example, the entire gene can be referred to as the target sequence for an assay, but amplification can generate copies of smaller sequences (e.g., about 85-200 nucleotides) contained in the target gene sequence. Known amplification methods include, for example, transcription-associated amplification, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), and strand displacement amplification (SDA). Replicase-mediated amplification uses self-replicating RNA molecules and a replicase, such as QB-replicase (U.S. Patent No. 4,786,600, Kramer et al.). PCR amplification uses DNA polymerase, primers, and temperature cycling to synthesize multiple copies of two complementary strands of DNA or cDNA (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Mullis et al.; and Methods in Enzymology, 1987, Vol. 155: 335-350). LCR amplification uses at least four separate oligonucleotides and multiple cycles. SDA amplifies the target and its complementary strand by hybridization, ligation and denaturation of the target strand (e.g., U.S. Pat. No. 5,427,930, Birkenmeyer et al.; U.S. Pat. No. 5,516,663, Backman et al.; and European Patent Application Publication No. 0320308). SDA uses a primer containing a recognition site for a restriction endonuclease, which allows the endonuclease to nick one strand of a semi-modified DNA duplex containing the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g., U.S. Pat. No. 5,422,252, Walker et al.; U.S. Pat. No. 5,547,861, Nadeau et al.; U.S. Pat. No. 5,648,211, Fraiser et al.). Transcription-associated amplification is a preferred embodiment described below. It will be apparent to one skilled in the art that the oligonucleotides and methods set forth in this preferred embodiment can be adapted by those skilled in the art of molecular biology for use in primer-dependent amplification systems.

[0030] "Transcription-associated amplification" or "transcription-mediated amplification" (TMA) is a method for the synthesis of nucleic acids using RNA polymerase. Any type of nucleic acid amplification method that produces multiple RNA transcripts from a nucleic acid template. Such methods generally use an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a template-complementary oligonucleotide that contains a promoter sequence and, optionally, one or more other oligonucleotides. Variations in transcription-associated amplification are well known to those skilled in the art, as previously reported (U.S. Pat. Nos. 5,399,491 and 5,554,516, Kacian et al.; U.S. Pat. No. 5,437,990, Burg et al.; International Publication Nos. WO88 / 01302 and WO88 / 10315, Gingeras et al.; U.S. Pat. No. 5,130,238, Malek et al.; U.S. Pat. Nos. 4,868,105 and 5,124,246, Urdea et al.; International Publication No. WO95 / 03430, Ryder et al.; and U.S. Patent Application No. 11 / 213,519, Becker et al., filed August 26, 2005). The TMA method of Kacian et al. is a preferred embodiment of the amplification method used to detect the target sequence described below. Although preferred embodiments are described using TMA, or transcription-associated amplification, one of skill in the art will recognize that the amplification oligomers disclosed herein can be readily adapted for use with other amplification methods based on polymerase-mediated extension of oligomer sequences.

[0031] "Detection probe" refers to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid, preferably in an amplified sequence, under conditions that promote hybridization, allowing detection of the target sequence or the amplified nucleic acid. Detection can be direct (i.e., by a probe that hybridizes directly to the target or amplified nucleic acid) or indirect (i.e., by a probe that hybridizes to an intermediate molecular structure that links the probe to the target or amplified nucleic acid). The "label" of the probe refers to a sequence that is generally within the amplified nucleic acid sequence (i.e., a subset) that specifically binds to at least a portion of the probe oligomer through standard hydrogen bonding (base pairing), although the probe sequence need not be 100% complementary to the target sequence. A sequence that is "sufficiently complementary" allows the probe oligomer to stably hybridize to a target sequence that is not completely complementary to the target specific sequence of the probe. In addition to the target specific sequence, the probe may contain other sequences that contribute to three-dimensional organization or detection function (e.g., U.S. Pat. Nos. 5,118,801 and 5,312,728, Lizardi et al.; U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945, Becker et al.).

[0032] "Sufficiently complementary" refers to a contiguous nucleic acid sequence capable of hybridizing to another sequence through hydrogen bonding between a series of complementary bases, which may be complementary at every position in the sequence through standard base pairing (e.g., G:C, A:T or A:U pairing) or may contain one or more non-complementary residues, so long as the entire sequence is capable of specifically hybridizing to another sequence under suitable hybridization conditions. The contiguous bases are preferably at least about 80%, more preferably at least about 90%, and most preferably about 100% complementary to the sequence to which an oligomer is intended to specifically hybridize. Suitable hybridization conditions are well known to those of skill in the art and can be readily estimated based on sequence composition or can be empirically determined by routine testing (see, e.g., Sambrooke et al., Molecular Cloning, A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring, NY)). Ring-Haber, 1989), §§ 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57, in particular §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

[0033] "Capture probe" or "capture oligomer" refers to at least one nucleic acid oligomer that provides a means for specifically binding a target sequence to a support in order to separate the target nucleic acid from other components in a mixture. The capture oligomer may be based on any known ligand interaction to link the label to the support, and preferred embodiments link the label to an oligomer immobilized on the support by base pair hybridization. Embodiments of the capture oligomer include two binding regions, a target sequence binding region and an immobilized probe binding region, usually on the same oligomer, although the regions may be on two different oligomers linked together by one or more linkers. "Immobilized probe" refers to a nucleic acid that directly or indirectly binds the capture oligomer to an immobilized support. The immobilized oligomer bound to a solid support facilitates separation of the bound target sequence from unbound material in the mixture. Any known solid support material can be used, such as nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene, and metal particles, preferably magnetically attachable particles, such as matrices or particles in solution. Preferred label capture materials and methods are as previously described (U.S. Patent Nos. 6,110,678 and 6,280,952, Weisburg et al.).

[0034] "Separation" or "purification" refers to the separation of one or more components in a biological sample from one or more other components in the biological sample. For example, sample components typically include, for example, proteins, carbohydrates, lipids, and nucleic acids in an aqueous solution that may contain organelles or cell debris. Preferably, the separation or purification step removes at least about 70%, more preferably at least about 90%, and even more preferably at least about 95% of other components present in the sample.

[0035] "Label" refers to a molecular moiety or compound that can be detected or that can produce a detectable response. The label can be directly or indirectly attached to the nucleic acid probe. Direct labeling can be achieved by a bond or interaction that links the label to the probe, including covalent or non-covalent interactions, such as hydrogen bonds, hydrophobic and ionic interactions, or the formation of chelate or coordination complexes. Indirect labeling can be achieved by using a directly or indirectly labeled bridging moiety, or "linker," that can enhance the detectable signal. The label can be any known detectable moiety, such as a radionuclide, a ligand, an enzyme or enzyme substrate, a reactive group, or a chromophore, such as a dye, bead or particle that imparts a detectable color, a luminescent compound (e.g., bioluminescent, phosphorescent, or chemiluminescent labels), and a fluorescent compound. Preferably, the label on the labeled probe can be detected in a homogeneous assay system, i.e., the bound labeled probe present in a mixture containing unbound probe exhibits a detectable change, such as, for example, stability or discernible degradation, compared to the unbound probe. Preferred labels for use in homogeneous assay systems are chemiluminescent compounds, with preferred chemiluminescent compounds being acridinium ester ("AE") compounds, including standard AE or derivatives thereof, such as naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE, 1- or 3-methyl-meta-difluoro-AE, and 2-methyl-AE (U.S. Patent Nos. 5,656,207, Woodhead et al.; 5,658,737, Nelson et al.; and 5,639,604, Arnold, Jr. et al.).Methods for synthesizing and detecting labels by attaching them to nucleic acids are well known (Sambrooke et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; US Pat. No. 5,331,631). Nos. 5,658,737, Nelson et al.; 5,656,207, Woodhead et al.; 5,547,842, Hogan et al.; 5,283,174, Arnold et al.; 4,581,333, Kourilsky et al.; and European Patent Application Publication No. O747706, Becker et al.).

[0036] "Consisting essentially of" means that the compositions, kits or methods of the invention may include one or more other components, compositions or steps that do not substantially alter the ability to detect multiple specific HPV target sequences of any one or all of groups A1, A2, C1, C2 and D by in vitro amplification reactions using the disclosed amplification oligomer embodiments and detection using the disclosed detection probe embodiments. The term does not include any one or more components, compositions or steps that prevent said materials or procedures from detecting multiple specific HPV target sequences of any one or all of groups A1, A2, C1, C2 and D by in vitro amplification and detection reactions.

[0037] The present invention includes compositions (nucleic acid capture oligomers, amplification oligomers and probes) and methods for detecting HPV nucleic acids in human biological samples. To select suitable oligomer sequences for use as capture probes, amplification oligomers and detection probes, known HPV RNA or DNA sequences (including partial sequences) of 31 known subtypes available from public databases (e.g., GenBank) were aligned by matching regions of identical or similar sequences in the E6 / E7 gene sequences and compared using well-known molecular biology methods. Other considerations in designing the oligomers include the relative GC content of the sequences, the relative absence of predicted secondary structures (e.g., hairpin turns that form intramolecular hybrids), and the relative absence of predicted intramolecular interactions when the oligomers are present in a mixture under hybridization conditions using methods well known in the art. This comparison and prediction can be facilitated using computerized algorithms, but one of skill in the art can easily perform the comparison manually. HPV E6 / E7 sequences were compared and sequences of closely related types with substantial sequence similarity were divided into groups A1, A2, B, C1, C2, and D. Sequence comparisons were performed both within and between closely related groups, and portions of the E6 / E7 sequences with relatively little sequence variation within the groups (i.e., consensus regions) were identified and selected as the basis for designing synthetic oligomers suitable for hybridization to target sequences in one or more HPV types. Designing oligomers to consensus sequences, rather than combinations of amplification and detection oligomers specific for each individual HPV type to be detected, reduces the total number of oligomers in the reaction mixture, reduces the probability of unintended oligomer interactions (e.g., formation of "primer dimers" or unlabeled amplification products) that are known to occur in amplification reactions, and also reduces background signal, which is generally directly proportional to the number of detection probes used in a single reaction. However, oligomers based on the use of consensus sequences are generally more difficult to design. The target regions available for designing consensus sequences are more limited because some of the designed oligomers may not be perfectly complementary to the intended target sequence or sequences.That is, for one or more HPV types to be detected using the oligomer combination, there may be one or more mismatches between the oligomer sequence and the complementary target sequence for which the oligomer is intended for one or more HPV types within the group. Alternatively, an oligomer may perfectly match the target sequence of one HPV type within a group (e.g., HPV16 within group A1) and contain one or more mismatches with other HPV types within the group (e.g., HPV31 and / or 35) yet still function in amplifying and / or detecting types within the group. Similarly, an oligomer designed to function for one group (e.g., an amplification oligomer for group A2) may function in combination with other amplification oligomers to amplify sequences of multiple types from more than one group (e.g., types from groups A1 and A2). Taking these into consideration, the amplification oligomer was designed to hybridize to the HPV gene sequence (sense strand or complementary strand sequence) in the E7 gene and amplify a sequence of generally about 80 to about 150 nucleotides (for example, in each embodiment, the following sequences were amplified: 107 nt of HPV16, 152 nt of HPV18, 118 nt of HPV31, 82 nt of HPV33, 109 nt of HPV35, 96 nt of HPV39, 152 nt of HPV45, 133 nt of HPV51, 86 nt of HPV52, 146 nt of HPV56, 83 to 85 nt of HPV58, 132 nt of HPV59, and 99 nt of HPV68). The detection probe was designed to detect the sequence between the target sequences of a particular pair of primers so as to hybridize to the target sequence contained in the sequence amplified using the corresponding primer pair. The target region for designing the capture probe oligomers was generally 5' to the target sequence for amplification of the HPV type or group in the E6 / E7 gene sequence. In many cases, iterative design and empirical testing was performed to optimize the selection of oligomers shown in Table 1.

[0038] The capture probe may be any of the following SEQ ID NOs: 1 to 10. Preferred embodiments of the capture probe include a substantially homopolymeric 3' tail region (T3A) as shown in SEQ ID NOs: 1, 3, 5, 7, and 9. 30 ) are some examples.

[0039] The detection probe oligomer may be any of SEQ ID NOs: 11 to 17, 41, 44 to 54, and 58. From about 50 designs that were synthesized and tested as oligomers, the preferred probe oligomer size is in the range of 20 to 23 nt, the GC content is about 40 to 50%, and the estimated melting temperature (T m) was about 55-70°C. Each embodiment of the probe was synthesized and labeled with a chemiluminescent AE compound (e.g., 2-methyl-AE) using a non-nucleotidic linker moiety and previously reported methods (U.S. Patent Nos. 5,185,439, 5,585,481, and 5,656,744, Arnold Jr. et al.; and U.S. Patent No. 5,639,604, Arnold et al., column 10, line 6 to column 11, line 3, and Example 8). Preferred locations for labeling are in the central region of the oligomer, near A / T base pair regions, at the 3' or 5' termini, or at or near mismatch locations where there are known non-target sequences to be avoided for detection. Embodiments of AE-labeled probes include those at the following positions: SEQ ID NOs: 15 and 53 between nt 4 and 5; SEQ ID NOs: 11, 14, 17, 44, 50 and 52 between nt 5 and 6; SEQ ID NOs: 15, 17, 47, 48, 52 and 54 between nt 7 and 8; SEQ ID NOs: 12, 14, 41, 45, 47, 49, 54 and 58 between nt 8 and 9; SEQ ID NOs: 47 and 48 between nt 9 and 10; SEQ ID NOs: 17, 44 and 47 between nt 10 and 11; SEQ ID NOs: 13, 44, 45, 46, 47, 50, 51 and 53 between nt 11 and 12; between nt 12 and 13 for SEQ ID NOs: 13, 15, 51 and 53; between nt 13 and 14 for SEQ ID NOs: 14, 16, 45, 47 and 54; between nt 14 and 15 for SEQ ID NOs: 13, 14, 49 and 52; between nt 15 and 16 for SEQ ID NOs: 13 and 52; between nt 16 and 17 for SEQ ID NOs: 44; between nt 17 and 18 for SEQ ID NOs: 44, 47 and 58; between nt 19 and 20 for SEQ ID NO: 51; between nt 20 and 21 for SEQ ID NO: 50; and between nt 21 and 22 for SEQ ID NO: 44. Preferred embodiments include AE-labeled probes labeled at the following positions: SEQ ID NO:11 between nt 5 and 6; SEQ ID NOs:15 and 17 between nt 7 and 8; SEQ ID NO:12 between nt 8 and 9; SEQ ID NO:44 between nt 10 and 11; SEQ ID NO:45 between nt 11 and 12; SEQ ID NO:14 between nt 13 and 14; and SEQ ID NO:52 between nt 14 and 15. The detection probes of the preferred embodiments were synthesized as RNA sequences linked with a 2'-methoxy backbone.The probe embodiments of SEQ ID NOs:49-54 cumulatively target the sequence of SEQ ID NO:55, and the overlapping probes of SEQ ID NOs:49-53 all contain the sequence of SEQ ID NO:56.

[0040] In an embodiment of the detection probe, a mixture of probes for detecting HPV sequences in a plurality of HPV types included in groups A1, A2, C2, C2, and D, i.e., HPV types 16, 31, 35, 33, 52, 58, 18, 45, 59, 39, 68, 51, and 56, is included. A preferred embodiment of the mixture is composed of detection probes of SEQ ID NOs: 11 to 15 and 17. A particularly preferred embodiment is a mixture of the following AE-labeled probes: SEQ ID NO: 11 labeled between nt 5 and 6; SEQ ID NO: 12 labeled between nt 8 and 9; SEQ ID NO: 13 labeled between nt 15 and 16; SEQ ID NO: 14 labeled between nt 13 and 14; SEQ ID NO: 15 labeled between nt 7 and 8; and SEQ ID NO: 17 labeled between nt 7 and 8. Another preferred embodiment of the mixture is composed of probes of SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45, and 52. A particularly preferred embodiment is a mixture of the following AE-labeled probes: SEQ ID NO:11 labeled between nt 5 and 6; SEQ ID NO:12 labeled between nt 8 and 9; SEQ ID NO:14 labeled between nt 13 and 14; SEQ ID NO:15 labeled between nt 7 and 8; SEQ ID NO:17 labeled between nt 7 and 8; SEQ ID NO:44 labeled between nt 10 and 11; SEQ ID NO:45 labeled between nt 11 and 12; and SEQ ID NO:52 labeled between nt 14 and 15. The skilled artisan will recognize that the detection probes can be used to detect HPVs of individual groups, e.g., combinations of HPV types within groups A1, A2, C1, C2 or D or related types, e.g., groups A1 and A2. Preferred embodiments of the detection probes identified by each of the above label groups include the following: SEQ ID NO:11 for group A1; SEQ ID NO:12 for group A2; SEQ ID NO:13 or a mixture of SEQ ID NOs:44, 45 and 52 for group C1; SEQ ID NO:14 for group C2; and SEQ ID NOs:15 and 17 for group D.

[0041] Embodiments of amplification oligomers include SEQ ID NOs: 18 to 42. Some embodiments contain only sequences targeting HPV sequences: SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 39, 40, 41, and 42, while others contain other functional sequences, i.e., promoter sequences. Embodiments of promoter primers containing the promoter sequence of SEQ ID NO: 43 at the 5' end of the oligomer include the sequences of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36. Structurally related sequences as promoter primers comprising HPV-specific sequences and a 5' promoter linked to the HPV-specific sequences are SEQ ID NOs: 19 and 18, 42 and 18, 21 and 20, 23 and 22, 25 and 24, 27 and 26, 29 and 28, 31 and 30, 33 and 32, 35 and 34, and 37 and 36. Preferred mixtures or combinations of amplification oligomers for amplifying groups of HPV type sequences include the following: Group A1, SEQ ID NOs: 18 or 19, 20 or 21, and 22 or 23, and SEQ ID NO: 38; Group A2, SEQ ID NOs: 24 or 25, and 26 or 27, and SEQ ID NO: 38; Group C1, SEQ ID NOs: 28 or 29, and 30 or 31, and SEQ ID NO: 39; Group C2, SEQ ID NOs: 32 or 33, and SEQ ID NO: 40; and Group D, SEQ ID NOs: 34 or 35, and 36 or 37, and SEQ ID NOs: A preferred embodiment uses an amplification oligomer mixture that amplifies sequences from HPV types 16, 31, 35, 33, 52, 58, 18, 45, 59, 39, 68, 51, and 56 (i.e., HPVs in all of groups A1, A2, C1, C2, and D). A preferred embodiment of the amplification oligomer mixture for HPV types 16, 31, 35, 33, 52, 58, 18, 45, 59, 39, 68, 51, and 56 includes SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 39, 40, and 41.Other preferred embodiments of the amplification oligomer mixture for HPV types 16, 31, 35, 33, 52, 58, 18, 45, 59, 39, 68, 51, and 56 include SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 39, 40, and 41.

[0042] An assay method for detecting multiple HPV sequences in a biological sample includes the steps of: amplifying at least one HPV nucleic acid sequence of the E6 / E7 target region from an HPV type included in HPV groups A1, A2, C1, C2, and D using at least two amplification oligomers as primers, preferably by a transcription-associated amplification reaction, to generate an amplified nucleic acid; detecting the amplified nucleic acid by hybridizing with a detection probe sequence that is sufficiently complementary to a sequence included in the amplified nucleic acid. In a preferred embodiment, an amplification oligomer mixture of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 39, 40, and 41 is used in the amplification reaction, i.e., the transcription-associated amplification reaction. In another preferred embodiment, an amplification oligomer mixture of SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 39, 40, and 41 is used in the amplification reaction. The amplified nucleic acid is detected by detecting a signal generated by specific binding of the detection probe to the amplified sequence, but the specific binding does not have to occur by hybridization of a sequence in the amplified nucleic acid that is completely complementary to the detection probe sequence. The signal may be detected when the amplified sequence is unlabeled and bound to a labeled detection probe, in which case the bound labeled detection probe generates a detectable signal. The signal may be detected by detecting a complex containing the amplified nucleic acid and the probe after the amplified sequence binds to an unlabeled detection probe, for example by detecting a signal, such as an electrical signal, generated by the formation of a hybridization complex composed of the detection probe and the amplified nucleic acid. In a preferred embodiment, a detection probe of SEQ ID NO: 11 to 17 labeled with a chemiluminescent compound is used. In a preferred embodiment, a detection probe mixture is used to detect amplified HPV sequences of HPV types 16, 31, 35, 33, 52, 58, 18, 45, 59, 39, 68, 51 and 56. A preferred embodiment of the probe mixture used for detection is composed of the probes of SEQ ID NOs: 11 to 15 and 17, more preferably those labeled with a chemiluminescent compound. Another preferred embodiment of the probe mixture used for detection is a mixture of AE-labeled probes of SEQ ID NOs: 11 to 15 and 17.Another preferred embodiment of the probe mixture for use in detection is comprised of probes of SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45 and 52, more preferably labeled with a chemiluminescent compound. Another preferred embodiment of the probe mixture for use in detection is an AE-labeled probe mixture of SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45 and 52. Another embodiment of the assay can use detection probes to detect HPV types of individual groups, such as in a confirmatory assay of amplified nucleic acid to detect HPV types of groups A1, A2, C1, C2 or D, or to detect combinations of HPV types, such as groups A1 and A2. In a preferred embodiment of the assay, the following detection probes are used: SEQ ID NO: 11 for detection of HPV types of group A1; SEQ ID NO: 12 for detection of HPV types of group A2; SEQ ID NO: 13 or a mixture of SEQ ID NOs: 44, 45 and 52 for detection of HPV types of group C1; SEQ ID NO: 14 for detection of HPV types of group C2; or SEQ ID NOs: 15 and 17 for detection of HPV types of group D.

[0043] Another embodiment of this assay uses two amplification and detection reactions for each biological sample being tested. The first reaction includes an amplification oligomer mixture capable of amplifying HPV types of groups A1, A2, C1, C2, or D to generate at least one amplified HPV type from at least one HPV type. The second reaction includes an amplification oligomer mixture that amplifies sequences of both HPV types 16 and 18. The amplification products of the two amplification reactions can then be detected separately. The first amplification reaction detects the amplification products with a detection probe mixture that detects HPV types of groups A1, A2, C1, C2, or D to detect the presence of at least one of said HPV types in the sample. The second reaction detects the amplification products with a detection probe mixture specific for detecting amplified sequences of HPV16 and HPV18 to detect the presence of HPV16, HPV18, or both types in the sample. This assay provides additional diagnostic information for the following reasons: If the first amplification and detection reaction gives a positive result, the sample contained one of the HPV types of groups A1, A2, C1, C2 or D. If the second amplification and detection reaction also gives a positive result, the test sample contained at least one of the most common HR-HPV types, HPV16 and / or HPV18. If the first reaction gives a positive result and the second reaction gives a negative result, an HR-HPV type is present in the sample, but not HPV16 or HPV18. Based on this information, medical personnel can select appropriate follow-up monitoring for the patient. In one embodiment of this assay, an amplification oligomer mixture of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 39, 40, and 41, or an amplification oligomer mixture of SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 39, 40, and 41 is used in the first reaction, and the amplification product is detected with a probe oligomer mixture of SEQ ID NOs: 11 to 15 and 17, or a mixture composed of probes SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45, and 52.The second reaction for detection of HPV16 and HPV18 uses an amplification oligomer mixture of SEQ ID NOs: 18, 28, and 39, or an amplification oligomer mixture of SEQ ID NOs: 19, 29, and 39 in the amplification step and a detection probe mixture of SEQ ID NOs: 11 and 13 in the detection step. Another single sample assay embodiment includes three reactions: a first reaction that amplifies and detects any one of the HPV types of groups A1, A2, C1, C2, or D as described above, a second reaction that amplifies and detects only HPV16 sequences, and a third reaction that amplifies and detects only HPV18 sequences. This three-part assay provides important diagnostic information regarding the types of HPV nucleic acid present in the sample using relatively few reactions.

[0044] The assay for HPV detection also includes a step of isolating or purifying the labeled HPV RNA using a capture step prior to the amplification step. In a preferred embodiment, a capture probe oligomer mixture is used under hybridization conditions. A portion of the capture oligomer specifically hybridizes to the HPVE6 / E7 target sequence of at least one HPV type of groups A1, A2, C1, C2, or D, and another portion of the capture oligomer acts as one member of a binding pair (ligand) that immobilizes the captured HPV RNA to a solid support to facilitate separation of the captured RNA from other sample components prior to the amplification step. One or more amplification oligomers for use in the subsequent amplification reaction may be included during the HPV capture step, since the amplification oligomer hybridizes to a target sequence in the HPV RNA that is distinct from the target sequence of the capture probe oligomer, and presents a primer on the target for initiation of the amplification reaction without interfering with the target capture. In a preferred embodiment, a capture probe mixture containing SEQ ID NOs: 2, 4, 6, 8, and 10 is used. In another preferred embodiment, a capture probe oligomer mixture is used that contains HPV-specific sequences of SEQ ID NOs: 2, 4, 6, 8 and 10 covalently linked to the 3' tail portion, as in the capture probe mixture of SEQ ID NOs: 1, 3, 5, 7 and 9. The HPV-specific sequence of the capture probe hybridizes to the HPV target sequence, and the tail portion hybridizes to a complementary sequence (oligo-dT) immobilized on a solid support. In order to take advantage of the hybridization kinetics associated with solution-phase nucleic acid hybridization, the hybridization of the capture step is preferably performed with the capture oligomer and HPV target RNA free in solution. This hybridization produces a capture oligomer:HPV RNA complex that then binds to the immobilized probe by hybridization of the tail portion of the capture oligomer with the immobilized complementary sequence, and the resulting complex is separated from other sample components by standard methods (e.g., filtration, centrifugation, magnetic separation). Those skilled in the art will appreciate that any binding pair ligand or complementary sequence pair between the tail portion and the immobilized probe can be used to perform the capture step.In a preferred embodiment, the immobilized probe is oligo-dT attached to a magnetically bindable monodisperse particle, so that the hybridization complex containing HPV RNA is easily separated from the solution by applying a magnetic force to the vessel containing the reaction mixture. The captured HPV labeled RNA is washed one or more times to further purify the label from potential inhibitors (e.g., the particles with the attached HPV RNA: capture oligomer: immobilized probe complex are resuspended in a washing solution, and the particles with the attached complex are recovered from the washing solution). To reduce the number of operation steps, the HPV labeled nucleic acid may be amplified without being released from the capture oligomer. If the amplification oligomer is present during the label capture step, the initial amplification reaction (cDNA formation) can be achieved by polymerization from the 3' end of the amplification oligomer hybridized to the captured HPV labeled RNA.

[0045] Assays for HPV detection may optionally include a non-HPV-internal control (IC) nucleic acid, which is amplified and detected in the same assay reaction mixture by amplification and detection oligomers specific for the IC sequence, giving a positive signal distinct from the HPV-related signal (e.g., a different AE label is used for the IC-specific probe compared to the HPV probe, and the distinguishable signal is detected separately by known methods (e.g., U.S. Pat. Nos. 5,658,737, 5,756,706, 5,827,656, and 5,840,873, Nelson et al.)). The detection of a signal from the amplified IC sequence proves that the assay reagents, conditions, and performance of the assay steps were properly used in the assay, in the absence of a signal for the HPV label (e.g., a sample showing a negative result for the presence of HPV). The IC can be used as an internal calibrator for the assay when a quantitative result is required. That is, the signal obtained from the amplification and detection of the IC is used to set the parameters used in the algorithm to quantify the amount of HPV nucleic acid in the sample based on the signal obtained for the amplified HPV target sequence. A preferred embodiment of the IC is a randomized sequence from a natural source (e.g., a randomly rearranged HPV sequence). A preferred IC is an in vitro synthesized RNA transcript, e.g., a transcript made from a cloned randomized sequence, to obtain an accurate IC amount per reaction. Primers and probes specific for the in vitro synthesized IC target sequence are used to amplify the IC target and detect the amplified IC sequence using assay conditions substantially identical to those used for amplifying and detecting the HPV sequence. In a preferred embodiment that includes a target capture step, the assay further includes an IC-specific capture probe in the same capture step as that used for purifying the HPV target. Thus, the IC is treated in the same manner as the HPV analyte in all steps of the assay.

[0046] Amplification of the captured HPV target region can be achieved using various known nucleic acid amplification reactions, but preferably using transcription-associated amplification reactions. In the above embodiment, multiple nucleic acid strands are generated from a single copy of the target nucleic acid, and thus the target can be detected by a detection probe that is bound to the amplified sequence. Transcription-associated amplification is performed under substantially isothermal conditions, as previously reported (e.g., transcription-mediated amplification (TMA) described by Kacian et al. in U.S. Pat. Nos. 5,399,491 and 5,554,516, and nucleic acid sequence-based amplification (NASBA) described by Davey et al. in U.S. Pat. No. 5,409,818). Briefly, transcription-associated amplification uses a promoter primer containing a target-specific sequence and a promoter sequence for an RNA polymerase, another primer, enzymes that provide synthetic and degradative activities (reverse transcriptase (RT), DNA-dependent RNase, and RNA polymerase), substrates (deoxyribonucleoside triphosphates, ribonucleoside triphosphates), and appropriate salts, buffers, and cofactors in solution to generate multiple RNA transcripts from a nucleic acid template. Briefly, the promoter primer hybridizes specifically to a target RNA sequence, and the reverse transcriptase extends from the 3' end of the promoter primer to form a first strand cDNA. This cDNA is available for hybridization with a second primer in any manner (e.g., denaturation of the double strand), but preferably uses RNase H activity (e.g., provided by the RT enzyme) to digest the RNA strand in the cDNA:RNA duplex. The second primer binds to this cDNA, and a new DNA strand is synthesized by RT from the 3' end of the second primer to form a double-stranded DNA with a functional promoter sequence at one end. RNA polymerase binds to the promoter sequence and transcribes multiple transcripts ("amplicons") that are used in subsequent steps of the amplification process, each of which acts as a template for a new round of replication, generating large amounts of amplified nucleic acid (approximately 100-3,000 copies are synthesized from each RNA template strand).

[0047] The promoter primer oligonucleotide includes a 5' promoter sequence that functions as a promoter when an appropriate RNA polymerase is bound, and a 3' sequence that specifically hybridizes to the target region sequence. A preferred embodiment is a T7 promoter sequence specific to T7 RNA polymerase. Preferred embodiments of the promoter primer include SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, and are used in combination with the primers of SEQ ID NOs: 38 to 41.

[0048] For transcription-mediated amplification of HPV sequences, HPV RNA is selected from, for example, SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, and cDNA is synthesized from the promoter primer by RT activity using HPV RNA as a template. A second primer, for example, one or more of SEQ ID NOs: 38-41, then hybridizes to this cDNA strand, and RT-mediated polymerization forms a DNA double strand to form a functional double-stranded promoter to which a specific RNA polymerase binds and transcribes an RNA transcript from the HPV sequence to generate an HPV amplification product. Repeating this hybridization, polymerization, and cDNA synthesis step produces an RNA transcript and thus an HPV tag-specific amplification product.

[0049] It will be appreciated that other in vitro nucleic acid amplification methods using amplification oligomers (e.g., PCR or SDA) can also be used to amplify HPV target sequences, which do not require promoter sequences on the primers. In the amplification methods, preferred amplification oligomer mixtures use first primer oligomers of SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37, and second primer oligomers of SEQ ID NOs: 38-41.

[0050] The detection step typically uses a mixture of HPV-specific probes to detect any one of the labeled HPV types that may be present in the sample. That is, a positive result is obtained if at least one probe specifically binds to an amplified HPV sequence (e.g., RNA transcript or amplicon). For assays to detect HPV types of groups A1, A2, C1, C2 and D, the detection step uses a mixture of detection probes, preferably a mixture of SEQ ID NOs: 11-15 and 17, or a mixture of SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45 and 52. In assays with two or three amplification and detection reactions, to detect the presence of any one of the HPV types of groups A1, A2, C1, C2 and D in one reaction and a subset of HPVs, such as HPV16 and / or HPV18 in the other reaction, a single probe or a mixture of probe subsets is used in the detection step of the subset of HPV types (e.g. SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 44 individually, or a mixture of SEQ ID NO: 11 and SEQ ID NOs: 13 and 44). In a preferred embodiment, a labeled probe is used that generates a signal that is detected without purifying bound probe from unbound probe (i.e., a homogeneous detection system).More preferably, the probe is labeled with an acridinium ester (AE) compound to generate a detectable chemiluminescent signal (U.S. Pat. Nos. 5,283,174 and 5,656,744, Arnold et al.; U.S. Pat. No. 5,658,737, Nelson et al.).

[0051] In the methods described herein, the capture oligomers, amplification oligomers and detection probe oligomers have specific sequences identified as useful for detecting polymorphic HPV tag sequences in the E6 / E7 gene region of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Multiple oligomers were designed that hybridize to more than one HPV type in a genetically related group by selecting a tag sequence common to multiple HPV types or by creating a consensus sequence that has a structural feature common to multiple HPV types but is not identical to all types (or even to one type) in the targeted group. Embodiments of the amplification oligomers are SEQ ID NOs: 18-42 and embodiments of the detection probe oligomers are SEQ ID NOs: 11-17, 41, 44-54 and 58. A probe labeled with a chemiluminescent compound can have another oligomer substantially complementary to the probe oligomer hybridized to it, stabilizing the labeled probe during storage. Exemplary probe protection oligomers are SEQ ID NOs: 59-64. Preferred capture probe oligomer embodiments include HPV target binding sequences of SEQ ID NOs: 2, 4, 6, 8, and 10, which can bind to any moiety (i.e., ligand, e.g., biotin or avidin) that acts as a binding partner to link the target region sequence to a retrievable solid phase. Exemplary capture probes include the 3' tail sequence dT3A 30 The oligomers may also contain tail sequences that hybridize to immobilized complementary oligomers, such as SEQ ID NOS: 1, 3, 5, 7, and 9, which contain: Although the oligomers are generally disclosed as contiguous DNA sequences, they may also contain RNA equivalents, or analogous sequences containing substitutions at one or more positions with base analogs (e.g., inosine) or synthetic purine and pyrimidine derivatives, such as P or K bases (Lin & Brown, 1989, Nucl. Acids Res. 17:10373-83; Lin & Brown, 1992, Nucl. Acids Res. 20: 5149-52) function in substantially the same manner. It will be appreciated that embodiments of oligomers that are linked to an RNA label may include a 2'-methoxy backbone at one or more of the linkages of the nucleic acid backbone. Preferred embodiments synthesized with a 2'-methoxy backbone generally employ an RNA base at the position linked by the 2'-methoxy backbone.

[0052] Embodiments of the invention include kits made up of various combinations of materials useful for carrying out steps to detect HPV nucleic acid in a sample. Preferred kits contain amplification oligonucleotides that act as primers for in vitro amplification of nucleic acid sequences of HPV types of groups A1, A2, C1, C2 and D. One example of a kit contains first and second amplification oligonucleotides that are complementary to opposing strands of an HPV target sequence and flank both ends of the HPV sequence to be amplified. Each embodiment contains an amplification oligomer that includes SEQ ID NO: 18 or 19, 20 or 21, 22 or 23, 24 or 25, 26 or 27, 28 or 29, 30 or 31, 32 or 33, 34 or 35, and 36 or 37 as a first amplification oligomer and SEQ ID NO: 38-41 as a second primer. The first amplification oligonucleotide is preferably up to 100 nt in length, more preferably 20-60 nt, and may include one or more base analogue substitutions in said nucleotide bases complementary to the HPV target region sequence. The second amplification oligonucleotide is preferably up to 100 nt in length, more preferably 15-25 nt, and may include one or more nitrogenous base analogues. The kit embodiment may further contain one or more probe oligomers for detecting HPV amplification products. The probe embodiments include oligomers of SEQ ID NOs: 11-17, 44-54 and 58, preferably probes of SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45 and 52. The kit embodiment may further contain other oligomers, for example SEQ ID NOs: 57 and 59-64, loaded in a mixture with the probe oligomer to stabilize the functional component of the probe oligomer prior to use. The kit may also contain an oligomer that acts as a capture oligomer for purifying labeled nucleic acid from a sample. Examples of capture oligomers for use in the kit include oligomers containing about 25-30 nt complementary to an HPV tag sequence in the HPVE6 / E7 gene region in HPV types of groups A1, A2, C1, C2, and D. Preferred embodiments of the capture oligomer contained in the kit include oligomers containing the HPV-specific sequences of SEQ ID NOs: 2, 4, 6, 8, and 10 attached to a ligand that binds the capture oligomer to a solid support.A preferred embodiment of the capture oligomer in the kit includes a mixture of oligomers of SEQ ID NOs: 1, 3, 5, 7 and 9, which contain a substantially homopolymeric tail sequence attached to the 3' end of the HPV-specific sequence of SEQ ID NOs: 2, 4, 6, 8 and 10. It will be understood that the above kit embodiments include kits in which the oligomers are complementary sequences of the specified sequences, RNA and DNA equivalents, or RNA / DNA chimeras of the specified sequences, or substantially equivalent sequences which may contain one or more nucleotide analogue substitutions in the specified sequences. It will also be understood that the kit embodiments can contain the oligomers as individually packaged components, but preferably can contain mixtures of oligomers, such as a mixture of a labeled capture oligomer and a first amplification oligomer, or a mixture of probe oligomers. Kits useful for carrying out the methods of the invention include any of the amplification oligomers and / or detection probes disclosed herein packaged together. The kits may contain capture oligomers which can be packaged together with the amplification oligonucleotides and / or detection probes. Kits useful for carrying out the methods of the invention can further contain one or more reagents for use in the steps of label capture, in vitro amplification, and / or detection of amplification products as disclosed herein. It will be apparent to one of skill in the art that the invention encompasses a variety of kit formats.

[0053] The label capture step captures HPV RNA from the sample and catalyzes the transcription of the captured HPV label sequence. The oligomers described herein were tested in an assay method that involves amplification in an amplification reaction, detection of the amplified sequences in a homogeneous reaction with a labeled detection probe, and detection of a chemiluminescent signal generated by the bound probe that indicates the presence of HPV in a test sample. The general steps used in the assay have been previously reported (U.S. Patent Nos. 6,110,678, 6,280,952, and 6,534,273, Weisburg et al., relating to label capture; U.S. Patent Nos. 5,399,491 and 5,554,516, Kacian et al., relating to transcription-mediated amplification; and U.S. Patent Nos. 5,283,174 and 5,656,744, Arnold et al., and U.S. Patent No. 5,658,737, Nelson et al., relating to chemiluminescent labeling and detection).

[0054] The following reagents were generally used in the HPV detection assays described herein: Sample transport solution contained 15 mM sodium phosphate monobasic, 15 mM sodium phosphate dibasic, 1 mM EDTA, 1 mM EGTA, and 110 mM lithium lauryl sulfate (LLS), pH 6.7. Labeled Capture Reagents was dissolved in 250 mM HEPES, 310 mM lithium hydroxide, 1.88 M lithium chloride, 100 mM EDTA, pH 6.4, and 250 μg / ml magnetic particles (1 micron SERA-MAG™ MG-CM particles, Seradyn, Inc., Indianapolis, Ind.) containing (dT) 14 The oligomers were covalently linked. Cleaning solution contained 10 mM HEPES, 150 mM sodium chloride, 6.5 mM sodium hydroxide, 1 mM EDTA, 0.3% (v / v) ethanol, 0.02% (w / v) methylparaben, 0.01% (w / v) propylparaben, and 0.1% (w / v) sodium lauryl sulfate (pH 7.5). Probe Reagents contained one or more labeled detection probes in a solution consisting of 100 mM lithium succinate, 2% (w / v) LLS, 15 mM mercaptoethanesulfonate, 1.2 M lithium chloride, 20 mM EDTA, and 3% (v / v) ethanol (pH 4.7). Amplification Reagents was mixed with other reaction components to produce 47.6 mM Na-HEPES, 12.5 mM Mn-acetyl-L-cysteine, 2.5% TRITON™ X-100, 54.8 mM KCl, 23 mM MgCl2, 3 mM NaOH, 0.35 mM each of dNTPs (dATP, dCTP, dGTP, dTTP), 7.06 mM rATP, 1.35 mM rCTP, 1.35 mM MUT, 8.85 mM GTP, 0. 26mM Na2EDTA, 5% v / v glycerol, 2.9% trehalose, 0. A concentrated mixture results in a mixture containing 225% ethanol, 0.075% methylparaben, 0.015% propylparaben, and 0.002% phenol red (pH 7.5-7.6), although other formulations of the amplification reagent may work equally well. Primers may be added to the amplification reagent or may be added to the amplification reaction separately from the amplification reagent. enzyme Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) and bacteriophage T7 RNA polymerase, The units are functionally defined as follows: 1 UMMLV-RT incorporates 1 nmol TTP using 200-400 μmol oligo dT-primed poly(A) as template in 10 min at 37°C, and 1 UT7 RNA polymerase incorporates 1 nmol ATP into RNA using a DNA template containing a T7 promoter in 1 h at 37°C. Hybridization Reagents The buffer consisted of 100 mM succinic acid, 2% (w / v) LLS, 100 mM lithium hydroxide, 15 mM aldrithiol-2, 1.2 M lithium chloride, 20 mM EDTA, and 3.0% (v / v) ethanol (pH 4.7). Selection Reagent The buffer was 600 mM boric acid, 182.5 mM sodium hydroxide, 1% (v / v) octoxynol (TRITON® X-100), pH 8.5. Detection Reagents contains 1 mM nitric acid and 32 mM hydrogen peroxide Detection Reagent I and Detection Reagent IIwas 1.5M sodium hydroxide. The oligomers for the HPV assay were typically used at the following concentrations: 2.5 pmol per reaction for each capture probe oligomer, 2.5 pmol per reaction for each promoter primer in the labeled capture reagent, 15 pmol per reaction for each non-promoter primer in the amplification mixture, 1.25-7.5 pmol per reaction for each promoter primer in the amplification mixture, and 0.03 pmol per reaction for each detection probe. In the tests performed, the HPV markers were typically in vitro synthesized transcripts that contained all or part of the E6 / E7 gene sequence of the HPV type(s) to be detected, and were used at a concentration of 25-100,000 copies per reaction, usually 100 or 1000 copies per reaction. Further details are provided in the Examples below. The foregoing are representative of some embodiments of the invention. EXAMPLES

[0055] Target capture, amplification and detection of HPV E6 / E7 sequences This example describes the assay steps and conditions used in a number of tests to detect target sequences from HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 in a sample.

[0056] For the label capture step, a mixture of oligomers SEQ ID NOs: 1, 3, 5, 7 and 9 (2.5 pmol each) was used in a reaction containing 0.5 ml of label capture reagent containing 100 or 1000 copies of HPV labeled RNA (i.e., one or more E6 / E7 transcripts of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) appropriate for each test. The reaction mixture also contained 2.5 pmol each of oligomers of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36. The mixture was incubated at 62°C for 30 minutes and then at room temperature for 30 minutes to allow the oligomers to hybridize to the labeled sequences, then magnetic force was applied to the outside of the container (for approximately 5 to 10 minutes) to separate the hybridization complexes on the magnetic particles from the other reaction components, the other sample components were aspirated off, the hybridization complexes on the particles were washed at room temperature with 1 ml of washing solution, and magnetic force was applied and aspirated as above to separate the hybridization complexes on the magnetic particles from the washing solution.

[0057] The HPV-labeled RNA captured in hybridization complexes containing at least one T7 promoter primer was then used in amplification reactions. The reactions contained 40 mM Trizma base, pH 7.5, 17.5 mM KCl, 20 mM MgCl2, 5% polyvinylpyrrolidone (PVP), 1 mM dNTP, 4 mM rNTP, and 15 pmol of each SEQ ID NO:38-41 oligomer per reaction, as well as optimal amounts of the following T7 primer oligomers for each reaction: SEQ ID NO:18 (1.25 pmol), SEQ ID NO:20 (7.5 pmol), SEQ ID NO:22 (1.25 pmol), SEQ ID NO:24 (2.5 pmol), SEQ ID NO:26 (2.5 pmol), SEQ ID NO:28 (2.5 pmol), SEQ ID NO:30 (2.5 pmol), SEQ ID NO:32 (7.5 pmol), SEQ ID NO:34 (1.25 pmol), and SEQ ID NO:36 (1.25 pmol). The reactions were covered with a layer of inert oil (200 μl) to prevent evaporation and incubated for 10-15 min at 62° C., followed by 3-5 min at 42° C. Enzymes (approximately 750 U MLVRT and approximately 2000 U of T7 RNA polymerase per reaction) were then added, mixed, and the amplification reactions were incubated at 42° C. for approximately 1 h.

[0058] To detect the amplified sequences, 0.1 ml of probe reagent containing 0.03 pmol per reaction of each of the oligomer mixtures of SEQ ID NOs: 11 to 16 or the oligomer mixtures of SEQ ID NOs: 11 to 15 and 17 labeled with AE compounds was added to the amplification mixture, incubated at 62°C for about 20 minutes, followed by addition of 0.25 ml of selection reagent, and incubating the mixture at 62°C for about 10 minutes, then cooling the reaction to room temperature (about 15 minutes), adding detection reagents I and II in sequence, and then detecting the bound probes substantially as previously reported (U.S. Pat. No. 5,658,737, column 25, lines 27-46; Nelsonet al., 1996, Biochem. 35: 8429-8438, 8432). Chemiluminescence was generated from the luminometer and the signal (relative light units, or RLU) was measured using a luminometer (LeaderHC+, Gen-Probe Inc., San Diego, CA, USA). was detected (for 2 seconds).

[0059] In a series of multiplex assays, HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 were amplified and detected using the assay method described above using samples containing known amounts of each label (25-1000 copies per reaction). All of these HPV types generally produced positive signals ranging from about 650,000 RLU to over 2,000,000 RLU for samples containing 200 or more copies of each labeled HPV RNA, although HPV types 31, 35, 18, 45, 59, and 68 produced positive signals (over 500,000 RLU) even when only 25 copies of labeled RNA were present in the sample. The results showed that the approximate sensitivity of the multiplex assay was 25 copies per reaction for HPV18, 31, 35, 45, 51, 56, 59, and 68, 50 copies per reaction for HPV16, 200 copies per reaction for HPV33, 39, and 58, and 400 copies per reaction for HPV52.

[0060] To demonstrate that this multiplex assay does not cross-react with HPV types not intended to be detected by the assay, i.e. low-risk HPV types, the same multiplex amplification and detection reaction steps were performed using in vitro E6 / E7 transcripts prepared from low-risk HPV types 6, 11, 42, 43 and 44 as labeled nucleic acids (using 1,000,000 and 10,000,000 copies per reaction). That is, low-risk HPV types were tested at 1,000-10,000 times more copies per reaction than the high-risk HPV types of groups A1, A2, C1, C2 and D detected by this assay. The low-risk HPV labels tested in this assay gave negative results compared to the positive controls (1000 copies of HPV16, 18, 33, 39 and 51 in vitro E6 / E7 transcripts were tested in individual reactions for each labeled HPV). Positive controls produced positive signals of >1,000,000 RLU to >2,000,000 RLU, whereas low-risk HPV types produced negative signals (<approximately 14,000 RLU). EXAMPLES

[0061] Amplification and detection of HPV types of groups A2, C1, C2 and D This example shows several tests in which different groups of individual HPV types were amplified and detected using combinations of amplification and detection oligomers. Reactions were performed essentially as described above, using only the amplification and detection steps, in order to efficiently detect the target sequence in test samples prepared with known amounts of in vitro transcripts of the E7 region (100-100,000 copies per reaction). Negative controls were samples that were run identically but without the target transcript. Amplification reactions were performed for each group using the same amounts per reaction of each non-promoter primer (15 pmol) and each promoter primer (5-7.5 pmol) (shown by their sequence numbers with the data table below) tested in the group of reactions. Prepared samples containing known amounts of each HPV labeled RNA were mixed with amplification reagents (40 mM Trizma base, pH 7.5, 17.5 mM KCl, 20 mM MgCl2, 5% polyvinylpyrrolidone (PVP), 1 mM each dNTP, 4 mM each rNTP) and each amplification oligomer shown in the data table below for each group. The reactions were covered with a layer of inert oil to prevent evaporation and incubated at 62°C for 10-15 minutes, then at 42°C for 3-5 minutes, then enzymes (approximately 750 U MLVRT and approximately 2000 U of T7 RNA polymerase per reaction) were added, mixed, and the amplification reactions were incubated at 42°C for approximately 1 hour. After the amplification reaction, an aliquot of each amplification reaction was mixed with the hybridization reagent and the AE-labeled probe (100 fmol per reaction) shown in the data table below for each group, the mixture was incubated to allow the probe to hybridize to the amplification product (62°C for about 20 minutes), and then the selection reagent was added to inactivate the label on the unbound probe (62°C for about 10 minutes). The detection reaction mixture was cooled to room temperature, and detection reagents I and II were added in sequence to induce chemiluminescence from the bound probe, and the chemiluminescent signal (RLU) was measured substantially as previously reported (U.S. Patent No. 5,658,737; Nelsonet al., 1996, Biochem. 35: 8429-8438, 8432). ) was detected by a luminometer. The test results are shown below.

[0062] [Table 4]

[0063] [Table 5]

[0064] The results show that the selected oligomers were effective in amplifying and detecting the different HPV types within the group, with positive results being obtained in many cases with as few as 100 copies of labeled RNA per reaction. Numerous similar tests were performed using other amplification and detection oligomer combinations disclosed herein with each HPV labeled transcript, with positive results being obtained in many cases with as few as 100 copies of labeled RNA per reaction, and consistently with 1000 copies per reaction. EXAMPLES

[0065] A multiplex assay for amplifying and detecting HPV types of groups A1, A2, C1, C2, and D This example shows results obtained with a multiplex assay similar to that described in Example 1 that detected HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. This multiplex assay additionally contained an internal control (IC) RNA (a randomized non-HPV RNA sequence) that was simultaneously amplified and detected with IC-specific primers and probes under the same assay conditions used to detect the HPV analytes, generating distinguishable chemiluminescent signals.

[0066] In the labeled capture portion of the assay, a mixture of capture oligomers of SEQ ID NO:1 (0.65 pmol / μl), SEQ ID NO:3 and SEQ ID NO:9 (2.5 pmol / μl each) and SEQ ID NO:5 and SEQ ID NO:7 (0.5 pmol / μl each) was used in a reaction containing a labeled capture reagent and a sample containing a known amount of HPV labeled RNA (in vitro transcripts of the E6 / E7 gene region of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68; 100-1,000 copies per reaction). The capture reaction mixture was incubated at 62°C for 30 minutes and at room temperature for 30 minutes, sequentially, to form hybridization complexes composed of HPV RNA: capture oligomer: immobilized probe on the solid support particles. A magnetic force was applied to the outside of the container as above to separate the hybridization complexes on the particles from other sample components, the other sample components were aspirated off, and the hybridization complexes on the particles were subsequently washed.

[0067] The HPV-labeled RNA captured in the hybridization complex was then amplified in a transcription-mediated amplification reaction containing amplification reagents and amplification oligomers (provided to the reaction from a promoter primer mix and a non-promoter primer mix). The promoter primers used were SEQ ID NO:18 (1.25 pmol / μl), SEQ ID NO:20 (7.5 pmol / μl), SEQ ID NO:22 (1.25 pmol / μl), SEQ ID NO:24 (7.5 pmol / μl), SEQ ID NO:26 (7.5 pmol / μl), SEQ ID NO:28 (2.5 pmol / μl), SEQ ID NO:30 (2.5 pmol / μl), SEQ ID NO:32 (7.5 pmol / μl), SEQ ID NO:34 (1.25 pmol / μl), and SEQ ID NO:36 (1.25 pmol / μl). The other primers were SEQ ID NO:38, 39, 40, and 41 (15 pmol / μl each). A promoter primer and primer specific for the internal control were similarly added to the reaction containing the internal control RNA. The mixture was covered with a layer of inert oil to prevent evaporation and incubated at 62°C for 10-15 minutes, followed by approximately 5 minutes at 42°C. Enzymes (MMLVRT and T7 RNA polymerase) were added and mixed essentially as above, and the amplification reaction was incubated at 42°C for approximately 60 minutes (45-75 minutes).

[0068] To detect the amplified HPV sequences, aliquots of the amplification mixture were diluted with probe reagent and SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45, and 52 (each labeled with 2-methyl-AE, 1.5×10 per reaction). 6 The amplified internal control sequences were detected using a synthetic oligomer specific for the internal control, labeled with a distinguishable AE compound (2'-fluoro-AE), at 8.5 x 10 per reaction. 5 The detection mixture was incubated at 62° C. for about 20 minutes, then cooled to room temperature for about 5 minutes, the selection reagent was added, the mixture was incubated at 62° C. for about 10 minutes, then cooled to room temperature for about 15 minutes, at which point chemiluminescence was generated using detection reagents I and II, in order, and the chemiluminescence signal (RLU) generated from the AE label in the bound probe was detected substantially as described above.

[0069] A total of 10 replicate samples for each HPV label were tested in the assay and the copy number was assayed. In this regard, representative data is shown below for two different lots of amplification reagents used. For each type of replicate reaction, the average detection RLU (mean) for the amplified labeled HPV is shown, along with the % of tests considered positive if the detection RLU exceeded the background level obtained in identically treated samples that did not contain HPV labeled RNA. The results show that the multiplex assay detected HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, although there was some variability in the results (e.g., between individual specimens, individual assay performance or reagent lots). The results also show the relative sensitivity of the assay for the different tested HPV types.

[0070] [Table 6]

[0071] [Table 7]

[0072] The above examples illustrate aspects and preferred embodiments of the invention as defined in the claims. An amplification oligomer mixture, wherein each oligomer sequence in the mixture is selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 42, which includes complementary oligomer sequences or RNA equivalents of said specific sequences, as described below: a first amplification oligomer of SEQ ID NO: 19 or 42, 21, 23, 25, 27, 29, 31, 33 and 35, and a second amplification oligomer of SEQ ID NO: 37, 38, 39, 40 and 41, or a complementary oligomeric sequence or RNA equivalent of said oligomeric sequences; First amplification oligomers of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36, and second amplification oligomers of SEQ ID NOs: 37, 38, 39, 40 and 41, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; A first amplification oligomer of SEQ ID NO: 19 or 42, 21, 23, 25, 27, 29, 31, 33 and 35, and a second amplification oligomer of SEQ ID NO: 38, 39, 40 and 41, or a complementary oligomeric sequence or RNA equivalent of said oligomeric sequences; or First amplification oligomers of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36, and second amplification oligomers of SEQ ID NOs: 38, 39, 40 and 41, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; A mixture comprising: [Item 2] 2. The oligomer mixture of item 1, wherein one or more individual oligomers comprise a backbone comprising at least one 2'-methoxy RNA group, at least one 2'-fluoro substituted RNA group, at least one peptide nucleic acid linkage, at least one phosphorothioate linkage, at least one methylphosphonate linkage, or any combination thereof. [Item 3] The oligomer mixture of item 1 contained in the kit. [Item 4] An oligomer mixture, wherein each individual oligomer sequence in the mixture is selected from the group consisting of SEQ ID NO:11 to SEQ ID NO:17, SEQ ID NO:44 to SEQ ID NO:54, and SEQ ID NO:58, wherein the group includes complementary oligomer sequences or RNA equivalents of said specific sequences, as follows: SEQ ID NOs: 11-17, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; SEQ ID NOs: 11 to 15 and 17, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; Oligomers of SEQ ID NOs: 11 to 15 and 17, and at least one of SEQ ID NOs: 44 to 54 and SEQ ID NO: 58, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; or SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45 and 52, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; A mixture comprising: [Item 5] 5. The oligomer mixture of item 4, wherein each oligomer sequence comprises a label attached directly or indirectly to said oligomer. [Item 6] 5. The oligomer mixture of item 4, wherein each oligomer sequence comprises a label that is a chemiluminescent compound. [Item 7] 5. The oligomer mixture of item 4, wherein each oligomer sequence has a backbone that includes at least one 2'-methoxy RNA group. [Item 8] The oligomer mixture of item 4 contained in the kit. [Item 9] A mixture of at least two oligomers, each oligomer sequence in the mixture being selected from the group consisting of SEQ ID NOs: 1-10, the group including complementary oligomer sequences or RNA equivalents of said specific sequences, as described below: at least two oligomers selected from SEQ ID NOs: 2, 4, 6, 8, and 10, wherein a ligand moiety is attached to each oligomer; Oligomers of SEQ ID NOs: 2, 4, 6, 8 and 10, with a ligand moiety attached to each oligomer; At least two oligomers selected from SEQ ID NOs: 1, 3, 5, 7 and 9; or Oligomers of SEQ ID NOs: 1, 3, 5, 7 and 9; A mixture comprising: [Item 10] 10. The oligomer mixture of item 9, wherein at least one oligomer comprises a backbone comprising at least one 2'-methoxy RNA group, at least one 2'-fluoro substituted RNA group, at least one peptide nucleic acid linkage, at least one phosphorothioate linkage, or at least one methylphosphonate linkage. [Item 11] The oligomer mixture of item 9 contained in the kit. [Item 12] 1. A method for detecting human papillomavirus (HPV) nucleic acid present in a biological sample, comprising: contacting nucleic acid in a biological sample containing RNA of at least one of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 with a mixture of amplification oligomers that amplify HPV sequences within an E6 / E7 target region sequence, said mixture comprising: a first amplification oligomer of SEQ ID NO: 19 or 42, 21, 23, 25, 27, 29, 31, 33 and 35, and a second amplification oligomer of SEQ ID NO: 37, 38, 39, 40 and 41, or a complementary oligomeric sequence or RNA equivalent of said oligomeric sequences; First amplification oligomers of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36, and second amplification oligomers of SEQ ID NOs: 37, 38, 39, 40 and 41, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; A first amplification oligomer of SEQ ID NO: 19 or 42, 21, 23, 25, 27, 29, 31, 33 and 35, and a second amplification oligomer of SEQ ID NO: 38, 39, 40 and 41, or a complementary oligomeric sequence or RNA equivalent of said oligomeric sequences; or First amplification oligomers of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36, and second amplification oligomers of SEQ ID NOs: 38, 39, 40 and 41, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; Consists of; amplifying in vitro HPV sequences derived from the target region sequence in at least one HPV type using the amplification oligomers and a nucleic acid polymerase to generate an HPV amplification product; and detecting the amplification products with a detection probe oligomer that is sufficiently complementary to specifically hybridize to the HPV amplification products to indicate the presence of at least one of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 in the sample; The method includes: [Item 13] Additionally, the capture oligomer may be contacted with HPV RNA in the sample prior to the amplification step. 13. The method of claim 12, comprising the steps 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, and separating the complex comprising the capture oligomer and the HPV RNA from other components in the sample by [Item 14] 14. The method of claim 13, wherein the capture oligomer is present in a capture oligomer mixture comprised of at least two oligomers, each oligomer sequence in the mixture being selected from the group consisting of SEQ ID NOs: 1-10, said group including complementary oligomer sequences or RNA equivalents of said specific sequences. [Item 15] The capture oligomer is: at least two oligomers selected from SEQ ID NOs: 2, 4, 6, 8, and 10, wherein a ligand moiety is attached to each oligomer; Oligomers of SEQ ID NOs: 2, 4, 6, 8 and 10, with a ligand moiety attached to each oligomer; At least two oligomers selected from SEQ ID NOs: 1, 3, 5, 7 and 9; or Oligomers of SEQ ID NOs: 1, 3, 5, 7 and 9; 14. The method of claim 13, wherein the capture oligomer mixture is comprised of: [Item 16] 13. The method of item 12, wherein the amplification step uses an amplification method that is substantially isothermal. [Item 17] 13. The method of item 12, wherein the amplification step uses a transcription-associated amplification method. [Item 18] 13. The method of item 12, wherein the detection step uses a mixture of probe oligomers, at least one probe oligomer in the mixture specifically binds to an HPV amplification product to generate a signal indicative of the presence of at least one HPV type in the sample. [Item 19] The detection process is as follows: SEQ ID NOs: 11-17, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; SEQ ID NOs: 11 to 15 and 17, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; SEQ ID NOs: 11 to 15 and 17, and at least one of SEQ ID NOs: 44 to 54 and SEQ ID NO: 58, or the complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; or SEQ ID NOs: 11, 12, 14, 15, 17, 44, 45 and 52, or complementary oligomeric sequences or RNA equivalents of said oligomeric sequences; 20. The method of item 18, using a probe oligomer mixture consisting of: [Item 20] the contacting step further comprises introducing a non-HPV-internal control sequence into the sample; the amplifying step further comprises amplifying the non-HPV-internal control sequence to generate an amplified internal control sequence; and the detecting step further comprises detecting the amplified internal control sequence to generate a signal indicative of proper performance of the step; Item 12 method.

Claims

[Claim 1] The invention described in the present specification.

Images