# Patent

## Title
Set of probes for the detection and typing of 46 human papillomavirus mucosal types

## Abstract

We have developed a set of probes to detect and identify 46 types of mucosal human papillomaviruses (HPV). These probes recognize the variable region comprised between the 2 conserved regions of the published GP5+/GP6+ set of PCR primers. The 46 probes have been shown to hybridize, as intended, to the DNA derived from the following HPV types: 6, 11, 13, 16, 18, 26, 30, 31, 32, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91 and 97. The hybridization of each probe is specific for each type without any cross hybridization among types and it is sensitive enough to allow detection of PCR products for genotyping of HPV DNA contained in clinical samples.

## Background
PRIOR APPLICATION INFORMATION
The instant application claims the benefit of U.S. Provisional Patent Application 61/296,245 filed Jan. 19, 2010


FIELD OF THE INVENTION
The present invention relates to reagents and methods for genotyping human papillomaviruses (HPV). In particular, the present invention relates to testing clinical samples for the type of HPV infection using a multiplex assay based on PCR amplification and detection using microspheres.
BACKGROUND OF THE INVENTION
Human papillomaviruses cause ubiquitous infectious of the keratinised epithelia of the skin and of the mucosae. About 120 HPV types have been characterized so far, which differ in prevalence, epidemiology and clinical manifestations (de Villiers et al., 2004). In particular, mucosal types infect the keratinised epithelia of the genital, anal and oro-pharyngeal mucosae (Muñoz et al., 2003; Muñoz and Bosch, 1997; Van Ranst et al, 1992; Chan et al., 1995, D'Souza et al., 2007, Bosh et al., 2008). Mucosal HPVs are most commonly transmitted by sexual contact, and infect sexually active people with a very high prevalence. It is estimated that the lifetime incidence of HPV infection in women is 80% (Bekkers at al., 2004), and the overall prevalence of active infection worldwide varies form 1.4% to 25% (Clifford at al, 2005).
Although the vast majority of infections are benign and self-limiting, a subset of “high risk” HPV types have the potential to cause persistent infection that may progress to malignant transformation and invasive cancer (Muñoz et al., 2003). Cervical cancer is the most common HPV-associated malignancy and it is now clear that HPV is a necessary cause of virtually all cervical cancers (Bosch and Muñoz, 2002; zur Hausen, 2002, Bosch et al., 2002; Muñoz et al., 2003; Walboomers et al., 1999, Smith et al, 2007). HPV associated malignancies are also found in the anal canal (Melbye and Sprogel, 1991; Palefsky et al., 1991), vulva (Buscema et al., 1988), the penis (Gregoire et al., 1995; Iwasawa et al., 1993), oro-pharyngeal mucosae and other head and neck tissues (D'Souza, et al., 2007; Mork et al., 2001; Gillison et al., 2000; Syrjanen, 2005).
Since HPV infection is necessary for the development of virtually all cervical cancers, detection of high risk HPV types is being considered as a screening method for cervical cancer, alongside, or even in substitution of, traditional cytological screening using the Papanicolau methods (pap test), with the promise of improving the sensitivity and cost effectiveness of cervical cancer screening programs (Cuzik et al., 2008; Cuzick et al., 2003; Ronco et al., 2006; Schiffman et al., 2005; Kim et al., 2005; Davies et al., 2006; Mayrand et al., 2006; Cuzik et al., 2006).
Two type-specific HPV vaccines (Gardasil, from Merck-Frosst for types 16, 18, 6 and 11; Cervarix form Glaxo-Smith-Kline for types 16 and 18) have recently been developed and clinical trials have shown that they are extremely effective in preventing both persistent infection with HPV and the dysplastic changes in the cervical epithelium that lead to malignant transformation (Koutsky et al., 2002; Villa et al., 2005; Harper et al., 2004; Harper et al., 2006). However, since vaccines are type-specific it is important to know the distribution of the various HPV types in a population, as well as to have a surveillance system in place to monitor vaccine efficacy and unexpected shifts in the frequency of HPV types not covered by the vaccines.
It is therefore expected that the routine use of type-specific tests for HPV will become more widespread, outside their current use in epidemiological studies for research purposes.
Currently, typing of HPV requires amplification by various PCR methods, followed by detection of specific sequences using either direct sequencing of the PCR products, RFLP methods (many methods have been described in the literature, for example Lungo et al., 1992; Menzo et al., 2008, Nobre et al., 2008; Santiago et al., 2006), Southern blot or dot blot using specific probes (for example Gregoire et al., 1989; Josefsson et al., 1999), reverse line hybridization (Gravitt et al., 1998; Kleter et al., 1999; van der Brule et al., 2002; Melchers et al, 1999), DNA microarray methods (Min et al., 2006; Albrecht et al, 2006; Choi et al., 2003; Huang et al., 2004; Hwang eta la., 2003; Oh et al., 2004; Nuovo et al., 2008), and others (for example Nishiwaki et al., 2008; Dell'Atti, 2007; Gao et al., 2003; Gharizadeh et al., 2007; Han et al., 2006; Lee et al., 2005; Liu et al, 2003; Zhang et al, 2003). In particular, reverse line blot methods have been validated and have been used extensively for epidemiological studies. Two leading commercial genotyping methods, InnoLiPA (van Hamont, 2006) and Roche linear array (Coutlee et al., 2006), are based on the reverse hybridization technology. The Roche Linear Array genotyping kit as been approved by FDA and it is the leading commercial genotyping method. However, these methods are not suitable for high throughput testing and they rely on a subjective visual assessment of band intensity for determining the results.
The xMAP technology developed by Luminex (Austin, Tex., USA) is based on microspheres that can be produced in 100 different “colours” depending on they ratio of two spectrally distinct fluorophores coupled to the microspheres. The different colours can be recognized by flow cytometers and the different type of microspheres can be enumerated and analyzed for the presence of specific bound ligands. This technology has been the basis for a variety of multiplex assays for serology, genotyping and other analytical applications. A description of the Luminex technology and a list of publications can be found at the Luminex web site.
Each type of microsphere can be coupled with a specific ligand, e.g. with DNA probes specific for each type of HPV in this work, and mixed together to form a multiplex assay. The PCR products derived from HPV samples are labelled with biotin and mixed with the beads carrying the probes, so that HPV DNA will hybridize with the cognate probe. The flow cytometer will then sort the different “coloured” microspheres and determine which type carries the fluorescence due to the HPV DNA. The computer software driving the flow cytometer will indicate which beads are fluorescent, thereby identifying the HPV type(s) present in the sample. The advantages of this method is the low cost per assay, the possibility of automation for a high throughput assay, and the flexibility derived from the possibility of adding or removing types of microspheres depending on the need of the assay or on the discovery of new types. Several microsphere-based multiplex assay for HPV genotyping have been published. The method by Wallace et al. (2005) is a multiplex microsphere assay with probes for 45 mucosal HPV. However, formal validation was performed for only a few types and only 20 types were detected from clinical samples, without independent validation of the genotyping result. The method published by Oh et al. (2007) detects 15 HPV types and it has been validated against a 132 clinical samples. A 56 sample comparison with a DNA microarray genotyping method is also shown. The method, by Schmitt et al. (2006), has been carefully validated with HPV plasmids and clinical samples and covers the 22 most common mucosal HPV types. The method by Jiang et al. (2006) describes specific probes for 26 HPV mucosal types. Validation was performed with synthetic oligonucleotides complementary to the probes and with a limited number of clinical samples. A commercial method developed by Qiagen (Hilden, Germany) is able to type 18 HPV high-risk using a proprietary set of primers, followed by detection using a Luminex system. At least one study comparing this Luminex Qiagen test to a reverse line blot hybridization has been published (Seme et al., 2009).
Herein, we report the design of novel HPV type-specific probes and the development of a microsphere multiplex assay that can detect 46 different mucosal types in a single reaction. In addition the unique probe set, compared to the previous method we introduce 2 innovations: i) the use of longer probes (30 mers) to provide for a greater specificity for variants and closely related types; ii) the production of single stranded DNA products by selective digestion of the PCR products with exonuclease, which produces a greater signal to noise ratio, making a washing step unnecessary.
SUMMARY OF THE INVENTION
We have described a set of 46 DNA probes and a PCR amplification method for the detection of 46 mucosal HPV types using the Luminex xMAP technology. This technology uses a mixture of sortable microsphere coupled to the specific HPV probes, so that all the 46 types can be detected simultaneously in one reaction tube.
Our data shows that all the probes are sensitive and specific for the detection of the 46 HPV types, without cross-hybridization. This conclusion is supported by the use of reference DNA from the 46 types and an extensive validation using direct sequencing as a gold standard for the identification of the HPV types.
Amplified DNAs from at least 32 HPV types can be detected simultaneously and precisely by this Luminex method.
Comparison with a leading commercial HPV typing method, the Roche Linear Array, confirms that the NML Luminex method is suitable for the identification of HPV types in clinical samples containing 3 or less HPV types. However, the PCR amplification method is less efficient in amplifying DNA from samples with multiple infections containing 4 or more HPV types. This is a problem caused by the PCR amplification method and not by the set of probes or the Luminex detection system. The less efficient amplification in multiple infections is a significant problems for HPV types 52, 53, 61, 73 84 and 89 but not for the major oncogenic HPV types, which are most important in epidemiology and clinical practice.
When samples with 4 or more HPV types are excluded, detection by NML Luminex and Roche Linear array are equivalent. Therefore, use of the NML Luminex method on populations with high frequency of multiple infections (such as HIV patients, men who have sex with men or sex workers) will lead to an underestimation of the prevalence with certain types. On the contrary, use of the NML Luminex method on a general population of women, where the prevalence of infections with 4 or more types is negligible, will produce accurate prevalence results for most types.
The NML Luminex HPV genotyping method has the advantage of detecting almost all genital HPV types and of being very sensitive thanks to the nested PCR method. The Luminex xMAP technology allows for a very quick, hands-off reading of the samples and an objective computational interpretation of the results. Because our method has no washing steps or visual reading steps, it is easily amenable to automation.
According to a first aspect of the invention, there is provided a method of detecting and typing a human papillomavirus (HPV) type infection in a sample comprising:
a) providing a sample suspected of comprising at least one HPV type;
b) adding to the sample primers suitable for amplifying the L1 region of HPV;
c) incubating the sample under conditions suitable for DNA amplification;
d) adding at least one probe having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1-46, said probe binding to only one HPV type under hybridization conditions, each said at least one probe further comprising a unique tag;
e) incubating said probe and said sample under conditions suitable for hybridization; and
f) detecting hybridization of at least one said tagged probe.
According to a second aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID NOs: 1-46.
According to a third aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID NOs: 1, 2, 4 or 5.
According to a fifth aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID. NOs: 4, 5 and 17.
According to a sixth aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID NOs: 4, 5, 8, 10, 11, 12, 17, 18, 19, 22, 23, 24, 27 and 29.
According to a seventh aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID NOs: 6, 4, 5, 7, 8, 10, 11, 12, 17, 18, 19, 20, 22, 23, 24, 27, 28, 29, 30, 31, 34, 37, 40 and 46.
According to an eighth aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID NOs: 1, 2, 9, 13, 14, 15, 16, 21, 25, 26, 27, 28, 29, 30, 31, 33, 35, 36, 38, 39, 41, 42, 43, 44 and 45.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1—Preliminary hybridization tests using 20 mer probes for HPV 6 and HPV 16—A mixture of two types of microspheres coupled with 20 mer probes for HPV 6 and 16 (as described in Table 1) were hybridized with the indicated volume of PCR reaction (panel A and B), with a 20 mer oligonucleotide exactly complementary to the HPV 16 probe (panel C), or with an unrelated PCR product (β-globin DNA).
FIG. 2—Position of the probes for the 46 HPV types considered in this submission. The alignment of the L1 region comprised between the GP5+/GP6+ primers is shown. The positions of the primers in indicated by the boxes while the probe sequences are in bold and underlined. Shown are probes for HPV 6 (SEQ ID NO: 231), HPV 11 (SEQ ID NO: 232): HPV 13 (SEQ ID NO: 233), HPV 16 (SEQ ID NO: 234), HPV 18 (SEQ ID NO: 235), HPV 26 (SEQ ID NO: 236), HPV 30 (SEQ ID NO: 237), HPV 31 (SEQ ID NO: 238), HPV 32 (SEQ ID NO: 239), HPV 33 (SEQ ID NO: 240), HPV 35 (SEQ ID NO: 241), HPV 39 (SEQ ID NO: 242), HPV 40 (SEQ ID NO: 243), HPV 42 (SEQ ID NO: 244), HPV 43 (SEQ ID NO: 245), HPV 44 (SEQ ID NO: 246), HPV 45 (SEQ ID NO: 247), HPV 51 (SEQ ID NO: 248), HPV 52 (SEQ ID NO: 249), HPV 53 (SEQ ID NO: 250), HPV 54 (SEQ ID NO: 251), HPV 56 (SEQ ID NO: 252), HPV 58 (SEQ ID NO: 253), HPV 59 (SEQ ID NO: 254), HPV 61 (SEQ ID NO: 255), HPV 62 (SEQ ID NO: 256), HPV 66 (SEQ ID NO: 257), HPV 67 (SEQ ID NO: 258), HPV 68 (SEQ ID NO: 259), HPV 69 (SEQ ID NO: 260), HPV 70 (SEQ ID NO: 261), HPV 71 (SEQ ID NO: 262), HPV 72 (SEQ ID NO: 263), HPV 73 (SEQ ID NO: 264), HPV 74 (SEQ ID NO: 265), HPV 81 (SEQ ID NO: 266), HPV 82 (SEQ ID NO: 267), HPV 83 (SEQ ID NO: 268), HPV 84 (SEQ ID NO: 269), HPV 85 (SEQ ID NO: 270), HPV 86 (SEQ ID NO: 271), HPV 87 (SEQ ID NO: 272), HPV 89 (SEQ ID NO: 273), HPV 90 (SEQ ID NO: 274), HPV 91 (SEQ ID NO: 275) and HPV 97 (SEQ ID NO: 276).
FIG. 3—Effect of T7 exonuclease digestion of nested PCR products on hybridization to Luminex beads. HPV 16 DNA was amplified by MY09/My11 and GP5+/GP6+ nested PCR, as described above, and the products were digested with T7 exonuclease for the indicated times. After digestion, the PCR products were hybridized to Luminex beads carrying the HPV 16 probe and detected as described above. The GP6+ primer contained a 5′ biotin moiety, for detection by the Luminex technology, and phosphorothioate bonds in the first 4 nucleotides on the 5′, to protect this strand from the action of the T7 exonuclease. The black bar and the white bar represent the fluorescence signals of a negative sample and of a sample containing a biotylinated oligonucleotide complementary to the HPV 16 probe.
FIG. 4—Graphic representation of the data of Table 4—The probes are on the right axis and the HPV DNAs on the left axis. The vertical axis represents the fluorescence read for each microsphere carrying a specific HPV probe. The bars on the diagonal represent the hybridization of HPV DNA type with the intended cognate probe.
FIG. 5—Simultaneous detection of multiple HPV types—Samples containing DNA from increasing numbers of HPV types were prepared as described in the text and then detected by the NML Luminex method as described. The number of types in each sample is indicated in the leftmost column. The second column from the left indicates what additional HPV type was added to the mixture. A “+” sign indicates a positive result (over 50 FU). The asterisks indicate false positive or false negative results.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.
DNA probes comprising SEQ ID NOs: 1 through 46 were generated according to the specific sequences of 46 types of genital HPV, namely type 6 (SEQ ID NO: 1), 11 (SEQ ID NO: 2), 13 (SEQ ID NO: 3), 16 (SEQ ID NO: 4), 18 (SEQ ID NO: 5), 26 (SEQ ID NO: 6), 30 (SEQ ID NO: 7), 31 (SEQ ID NO: 8), 32 (SEQ ID NO: 9), 33 (SEQ ID NO: 10), 35 (SEQ ID NO: 11), 39 (SEQ ID NO: 12), 40 (SEQ ID NO: 13), 42 (SEQ ID NO: 14), 43 (SEQ ID NO: 15), 44 (SEQ ID NO: 16), 45 (SEQ ID NO: 17), 51 (SEQ ID NO: 18), 52 (SEQ ID NO: 19), 53 (SEQ ID NO: 20), 54 (SEQ ID NO: 21), 56 (SEQ ID NO: 22), 58 (SEQ ID NO: 23), 59 (SEQ ID NO: 24), 61 (SEQ ID NO: 25), 62 (SEQ ID NO: 26), 66 (SEQ ID NO: 27), 67 (SEQ ID NO: 28), 68 (SEQ ID NO: 29), 69 (SEQ ID NO: 30), 70 (SEQ ID NO: 31), 71 (SEQ ID NO: 32), 72 (SEQ ID NO: 33), 73 (SEQ ID NO: 34), 74 (SEQ ID NO: 35), 81 (SEQ ID NO: 36), 82 (SEQ ID NO: 37), 83 (SEQ ID NO: 38), 84 (SEQ ID NO: 39), 85 (SEQ ID NO 40), 86 (SEQ ID NO: 41), 87 (SEQ ID NO: 42), 89 (SEQ ID NO: 43), 90 (SEQ ID NO: 44), 91 (SEQ ID NO: 45) and 97 (SEQ ID NO: 46). In order to make each probe sensitive and specific, the probes were tested in a multiplex assay as described below. Probes that in these tests did not hybridize to the intended HPV type or that cross-hybridized to other types were re-designed, sometimes repeatedly, until all probes hybridized to unique HPV type DNA. Accordingly, each respective probe binds specifically to only one specific HPV genome or HPV type. The history of the probe design is shown in Table 2.
For the multiplex assay, each probe was conjugated to one of 46 types of fluorescent microspheres, each with different ratios of red and infrared fluorophores, according to the manufacturers instructions. The micropsheres produced by Luminex Corp. are colour coded with a combination of two fluorescence dyes into 100 different sets that can be recognized and counted by a flow cytometer using a red laser. The flow cytometer can also detect a reporter dye bound to any set of beads using a separate green laser. For this embodiment, 46 sets of beads were selected and each set was coupled to a unique 30mer oligonucleotide probe designed to hybridize sensitively and specifically to one of 46 types of genital HPV DNA, amplified as described below. The 46 sets of beads were mixed to constitute a multiplex reaction that could detect any combination of the 46 types of HPV DNA present in clinical specimens.
The probes were designed to amplify the region comprised between the PCR amplification primers GP5+/GP6+. This region is 141 bp long for HPV 16 (nucleotides 6624 to 6764, GenBank accession no. AF125673), but varies in length slightly depending on the HPV type. GP5+/GP6+ are general primers that amplify the DNA from most HPV types. Published primers sets MY09/MY11 and primer set PGMY are also general primers which amplify most genital HPV types. They are situated outside the GP+/GP6+ region and therefore they can be used for a nested PCR reaction with the GP5+/GP6+ primers, in order to improve the sensitivity and the spectrum of HPV types that can be amplified, especially when multiple types are present in the same sample.
Using these primers, HPV DNA from clinical samples was amplified and then treated with T7 exonuclease to produce a single stranded, biotin labelled DNA complementary to the probes coupled to the microspheres. The single stranded HPV DNA and the tagged microspheres were then co-incubated, so that the HPV DNA could bind to its cognate probe on the microspheres. Streptavidin conjugated to the fluorophore phycoerythrine was then added. Streptavidin binds tightly to biotin conferring phycoerythrine fluorescence to those microsphere that are bound to HPV DNA. The samples were then analyzed by flow cytometry which provided an analysis of the numbers of each type of bound microspheres and their level of phycoerythrine fluorescence. High phycoerythrine fluoresce on specific beads indicates the presence of HPV DNA of specific types.
In addition to the multiplex assay for 46 HPV types, the microspheres can be mixed in different combinations to test separately only for HPV types contained in vaccines (HPV type 6 (SEQ ID NO: 1), 11 (SEQ ID NO: 2), 16 (SEQ ID NO: 4) and 18 (SEQ ID NO: 5)), or to test for the most malignant HPV types (HPV types 16 (SEQ ID NO: 4), 18 (SEQ ID NO: 5) and 45 (SEQ ID NO: 17)), or for the most common HPV types (HPV type 16 (SEQ ID NO: 4), 18 (SEQ ID NO: 5), 31 (SEQ ID NO: 8), 33 (SEQ ID NO: 10), 35 (SEQ ID NO: 11), 39 (SEQ ID NO: 12), 45 (SEQ ID NO: 17), 51 (SEQ ID NO: 18), 52 (SEQ ID NO: 19), 56 (SEQ ID NO: 22), 58 (SEQ ID NO: 23), 59 (SEQ ID NO: 24), 66 (SEQ ID NO: 27) and 68 (SEQ ID NO: 29)), or to test for all oncogenic HPV types (HPV type 26 (SEQ ID NO: 6), 16 (SEQ ID NO: 4), 18 (SEQ ID NO: 5), 30 (SEQ ID NO: 7), 31 (SEQ ID NO: 8), 33 (SEQ ID NO: 10), 0.35 (SEQ ID NO: 11), 39 (SEQ ID NO: 12), 45 (SEQ ID NO: 17), 51 (SEQ ID NO: 18), 52 (SEQ ID NO: 19), 53 (SEQ ID NO: 20), 56 (SEQ ID NO: 22), 58 (SEQ ID NO: 23), 59 (SEQ ID NO: 24), 66 (SEQ ID NO: 27), 67 (SEQ ID NO: 28), 68 (SEQ ID NO: 29), 69 (SEQ ID NO: 30), 70 (SEQ ID NO: 31), 73 (SEQ ID NO: 34), 82 (SEQ ID NO: 37), 85 (SEQ ID NO: 40), and 97 (SEQ ID NO: 46)), or to test only for non-oncogenic (low risk) types (6 (SEQ ID NO: 1), 11 (SEQ ID NO: 2), 32 (SEQ ID NO: 9), 40 (SEQ ID NO: 13), 42 (SEQ ID NO: 14), 43 (SEQ ID NO: 15), 44 (SEQ ID NO: 16), 54 (SEQ ID NO: 21), 61 (SEQ ID NO: 25), 62 (SEQ ID NO: 26), 66 (SEQ ID NO: 27), 67 (SEQ ID NO: 28), 68 (SEQ ID NO: 29), 69 (SEQ ID NO: 30), 70 (SEQ ID NO: 31), 72 (SEQ ID NO: 33), 74 (SEQ ID NO: 35), 81 (SEQ ID NO: 36), 83 (SEQ ID NO: 38), 84 (SEQ ID NO: 39), 86 (SEQ ID NO: 41), 87 (SEQ ID NO: 42), 89 (SEQ ID NO: 43), 90 (SEQ ID NO: 44) and 91 (SEQ ID NO: 45)).
In accordance with a first embodiment of the present invention, there is provided a series of DNA probes that can be used in conjunction with DNA amplification techniques to genotype various strains of HPV.
In a second embodiment of the invention, the series of DNA probes that can be used in a multiplexed format assay to simultaneously detect multiple strains of HPV
In a third embodiment of the invention, the DNA probes can be used with other detection systems including Southern or Northern blots, reverse line blot hybridization, DNA microarray or ELISA, or other such systems as will be obvious to those skilled in the art.
According to an aspect of the invention, there is provided a method of detecting and typing a human papillomavirus (HPV) type infection in a sample comprising:
a) providing a sample suspected of comprising at least one HPV type;
b) adding to the sample primers suitable for amplifying the L1 region of HPV;
c) incubating the sample under conditions suitable for DNA amplification;
d) adding at least one probe having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1-46, said probe binding to only one
HPV type under hybridization conditions, each said at least one probe further comprising a unique tag;
e) incubating said probe and said sample under conditions suitable for hybridization; and
f) detecting hybridization of at least one said tagged probe.
As discussed herein, the sample may contain more than one HPV type and the ‘at least one probe’ may be a set of probes comprising or consisting of respective probes having nucleotide sequences as set forth in any one of SEQ ID NOs: 1-46 and a unique tag or identification tag which uniquely identifies the respective probe. For example, all probes having a nucleotide sequence as set forth according to SEQ ID NO: 1 will have the same tag as will all probes having a nucleotide sequence as set forth in SEQ ID NO: 2.
As discussed herein, the hybridization conditions are sufficiently stringent that the probe will bind only to the target DNA. For example, the hybridization conditions may be sufficiently stringent for hybridization of two strands to occur only if there is 15, 16, 17, 18, 19, 20 or more consecutive nucleotides having an exact match.
As will be appreciated by one of skill in the art, the probes consisting of nucleotide sequences as set forth in any one of SEQ ID NOs: 1-46 and a unique tag can be used together or in any sub-combination thereof in a multiplex assay to specifically type HPV types in a given sample. Specifically, because each probe has a unique tag associated therewith, hybridization of a respective probe to a DNA molecule within the sample indicates the presence of the corresponding HPV type in that sample. The probe set is unique in that the probes do not cross-hybridize, as discussed below.
In some embodiments, at least one probe may refer to a mixture of probes, each representative probe of said mixture having a nucleotide sequence as set forth in SEQ ID NOs: 1, 2, 4 or 5 or as set forth in SEQ ID NOs: 4, 5 or 17 or as set forth in SEQ ID NOs: 4, 5, 8, 10, 11, 12, 17, 18, 19, 22, 23, 24, 27 or 29 or as set forth in SEQ ID NOs: 6, 4, 5, 7, 8, 10, 11, 12, 17, 18, 19, 20, 22, 23, 24, 27, 28, 29, 30, 31, 34, 37, 40 or 46 or as set forth in SEQ ID NOs: 1, 2, 9, 13, 14, 15, 16, 21, 25, 26, 27, 28, 29, 30, 31, 33, 35, 36, 38, 39, 41, 42, 43, 44 or 45.
The unique tag is a combination of two fluorescent dyes.
The unique tag is a combination of different ratios of red and infra-red fluorophores, as discussed herein.
According to another aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID NOs: 1-46.
According to another aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID NOs: 1, 2, 4 or 5. As will be appreciated by one of skill in the art, additional probes having sequences as set forth in any one of SEQ ID NOs: 3 and 6-46 and any combination thereof may be added to the probe set.
According to another aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID. NOs: 4, 5 and 17.
According to another aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID NOs: 4, 5, 8, 10, 11, 12, 17, 18, 19, 22, 23, 24, 27 and 29.
According to another aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID NOs: 6, 4, 5, 7, 8, 10, 11, 12, 17, 18, 19, 20, 22, 23, 24, 27, 28, 29, 30, 31, 34, 37, 40 and 46.
According to another aspect of the invention, there is provided a set of probes for detection and typing human papilloma virus (HPV) types, each said probe of said set hybridizing to only one HPV type under hybridizing conditions, each said probe of said set consisting of a unique tag and a nucleotide sequence as set forth in one of SEQ ID NOs: 1, 2, 9, 13, 14, 15, 16, 21, 25, 26, 27, 28, 29, 30, 31, 33, 35, 36, 38, 39, 41, 42, 43, 44 and 45.
EXAMPLES
Oligonucleotides
Oligonucleotides were synthesized at the DNA Core Section of the National Microbiology Laboratory. The probes carried a 5′ C12 amino linker modification for coupling to the carboxyl group of the Luminex microspheres. The MY09, MY11, GP5+ and the modified GP6+ primer for the PCR amplification of HPV DNA, were purchased from Invitrogen (Burlington ON, Canada).
PCR amplification
HPV DNA from plasmid or clinical specimens was amplified by a nested PCR method using the MY09/MY11 primers for the first step (Manos et al., 1989) and GP5+/GP6+ primers for the second step (Roda Husman et al., 1995). For optimal amplification of clinical samples with multiple HPV types, PGMY primers were used for the first step (Gravitt et al, 2000). The GP6+ primer carried the following modification: i) a 5′ biotin label to be used as a ligand for the streptavidin-PE for detection of PCR products (See below); ii) the first 4 nucleotides on the 5′ end were linked by phosphorothioate bonds to confer resistance to the action of the bacteriophage T7 gene 6 exonuclease (See below and in the result section). PCR amplification was performed in 1×PCR Buffer (Invitrogen, Cat #10342-020) in the presence of 4 mM MgCl2, 200 μM of dNTP (Invitrogen, Cat#10297-018), 0.2 mM of each primer and 1.25 U of Amplitaq Gold polymerase (Applied Biosystem, Cat #4311816). The first round of nested PCR amplification with the MY09/MY11 primers started with a 5 min initial denaturation step at 94° C., followed by 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds and elongation at 72° C. for 60 seconds, followed by a 7 min final extension at 72° C. Amplification with PGMY primers was carried out for 40 cycles (denaturation at 94° C. for 30 sec, annealing 55° C. for 30 sec, elongation 72° C. for 30 sec) in the presence of 6 mM MgCl2, 200 μM dNTPs and 0.6 μM each of the 18 primers comprising the PGMY mixture (Gravitt et al., 2000). One to 5 (typically 2) μL of this reaction were used for the second round of amplification with GP5+/GP6+ primers under the following conditions: 5 min initial denaturation at 94° C., followed by 30 cycles of 94° C. for 30 seconds, 40° C. for 20 seconds and 72° C. for 30 seconds, followed by a 7 min final extension at 72° C. One-step PCR with GP5+/GP6+ primers was conducted under the following conditions: a 5 min initial denaturation at 94° C. followed by 30 cycles of 94° C. for 30 seconds, 40° C. for 20 seconds and 72° C. for 30 seconds followed by a 7 min final extension at 72° C.
Digestion of PCR Products with T7 Exonuclease
After PCR, the GP5+ strand complementary to the biotinylated strand, was removed by digestion with T7 genes exonuclease, a 5′-3′ processive exonuclease. The other strand was protected from the action of T7 exonuclease by the 4 phosphorothioate bonds on the 5′ (Nikiforov et al, 1994). This digestion produced a single stranded, biotin labelled DNA complementary to the probes coupled to the Luminex beads and it was performed by adding T7 exonuclease (New New England Biolabs, Cat# M0263L) to PCR products at a final concentration of 0.4 U/μl. The reaction was stopped by adding 0.5M EDTA at a final concentration of 12.5 μl of 0.5M EDTA.
Preparation of Microspheres
Microspheres labelled with different ratios of red and infrared fluorophores were purchased from Luminex (Austin, Tex., USA, Cat #L100-CXXX-01) and coupled to HPV type-specific probes carrying a 5′ amino modification that reacts with the carboxyl groups on the microspheres following the instruction of the manufacturer with minor modifications. Briefly, the microsphere stock (Luminex) was vortexed vigorously then an aliquot containing 5.0×106 microspheres from each set was placed in a separate 1.5 ml microfuge tube, resuspended in a in a sonicating water bath (Branson) and centrifuged at 14000×g for 2 min. The supernatant was removed and the microshperes were resuspended in 50 μl of 0.1 M 2-N-morpholinoethansulfonic acid (MES) (Sigma Cat #M-2933) at a pH of 4.5. Then 1 μl of a 1 mM solution of the appropriate type of amino substituted oligonucleotide was added to a different set of microspheres and 2.54 of a 10 mg/mL solution of 1-Ethyl-3-3-dimethylaminopropyl carbodiimide HCl (EDC) (Fisher Cat #22980) were added to each tube. The tubes were vortexed and, after an incubation of 30 min at RT in the dark, 2.5 μL of 10 mg/ml EDC were added to each tube and incubated in the dark for 30 minutes. After the second incubation period 1 ml of 0.02% Tween 20 (Sigma Cat # P-9416) was added and the tubes were centrifuged for 2 minutes at 14,000×g. The supernatant was removed and 1 ml of 0.1% SDS (sodium dodecyl sulfate) was added to the microsphere pellet, the tubes were vortexed and then microcentrifuged for 2 minute at 14,000×g. The supernatant was removed and the pellet was resuspended in 100 μl of TE. The microspheres coupled to the probes were stored in the dark at 4° C. for a maximum of 6 months.
Luminex Assay
For the Luminex assay typically 15 microspheres/ul of each set were mixed in a reaction mixture. Exonuclease-digested PCR products were placed in a 96 well PCR microplate (Fisher, Cat # CS006509) in a total volume of 17 ul and sealed with a 96 well sealing cover (Fisher, Cat # CS006555). The microplate was incubated at 95° C. for 10 minutes to denature the DNA and 33 μL of the microsphere mix was added. The samples were incubated at the hybridization temperature of for 10 min and, after addition of 25 μl of a 0.04 mg/ul solution of streptavidine-phycoerythrin (Invitrogen Cat # S-866) in 1× tetramethyl ammonium chloride (TMAC) (Sigma, Cat # T-3411) was added to the samples and incubated for 5 more minutes at 60° C. Samples were analyzed on a Luminex Liquid Chip 200 flow cytometer using the Luminex IS software. The analysis was carried out at 60° C. with a maximum volume of 50 μL of sample and a minimum count of 100 microspheres per type, with a setting of 8,300 and 16,500 for the lower and upper gate, respectively.
Example 1
Design and Selection of Probes
The probes were targeted at the region of the L1 gene comprised between the GP5+/GP6+ primers (Roda Husman et al., 1995). This is a relatively poorly conserved region bracketed by two conserved regions were the GP5+/GP6+ primers bind. The length of this segment varies slightly among different types and, for example, it is 141 bp long in HPV16, corresponding to nt 6624 to 6764 of the sequence published by Flores et al., 1999 (GenBank accession no. AF125673).
Previous literature on the use of Luminex Xmap technology for detecting DNA sequence typically reported the use of 20 nt long probes. We therefore designed first 20 nt long probes, using the ArrayDesigner computer software (Premier BioSoft International) (Table 1), but preliminary experiments with probes and DNA from HPV type 6 and 16 showed that these probes were not sensitive for the detection of HPV DNA under our conditions. As shown in FIG. 1, DNA amplified from HPV 6 and HPV 16 clones failed to hybridize to the microsphere carrying the cognate 20 mer probe (Pane A and B). A biotylinated oliginucleotide exactly complementary to the HPV 16 probe did produce a considerable fluorescence of the HPV 16 microsphere but it also non-specifically increased the fluorescence of the HPV 6 microsphere (panel C).
Therefore, the probes were then re-designed as 30mers by adding 10 nt to the left or the right of the original probe. Longer probes also provide greater specificity and a better chance of discriminating among closely related HPV types or variants, for example HPV16 and HPV 31. This initial set or 30mers contained numerous unsuitable probes, either because they were cross-reactive (poor specificity) or because they were not binding efficiently to the intended target (poor sensitivity), or both. Unsuitable probes were redesigned typically by shifting their position 10 nucleotides to the right or to the left along the variable region of the GP5+/GP6+ fragment. This process was repeated until all probes were both specific and sensitive for the intended target. Attempts to predict the efficiency and specificity of the probes or to weed out probes with hairpins or other cross-reactive sequences proved ineffectual, because often probes behaved in an unexpected manner.
The history of the development of the probes is shown of Table 2, while the final sets of probes used for this method is shown on Table 3. FIG. 2 shows the location of the probes on the aligned sequences of the 46 HPV types covered by this method.
Example 2
Effect of Exonuclease
Simple denaturation of the double-stranded PCR products followed by hybridization to the probes on the microspheres produced a fluorescence signal that was much lower compared to the signal produced by hybridizing the microspheres to biotin-labelled single-stranded oligonucleotides (FIG. 3). We suspected that rehybridization of the long strands of the PCR products might have been thermodynamically more favourable than the hybridization of the GP6+ strand to the short (30 nt) probe physically constrained on the microsphere. We therefore decided to remove the non-labelled strand of the PCR product using bacteriophage T7 gene 6 exonuclease, according to the method described earlier (Nikiforov et al., 1994). T7 exonulease is a 5′→3′ processive enzyme that rapidly degrades one of the strand on a duplex DNA molecule (Kerr and Sadowski, 1972). In order to protect the GP6+ strand, carrying the biotin label, and selectively digest only GP5+ strand, the first 4 nucleotides at the 5′ end of the molecule were modified to include phosphorothioate bonds between the deoxyribose moieties, instead of the usual phosphodiester bonds. This chemical modification is known to inhibit the action of T7 exonuclease, that can no longer digest the DNA molecule starting from such modified end (Nikiforov et al., 1994).
Optimal digestion conditions were determined by incubating 40 units of T7 exonuclease with 100 ul of PCR product for various times, and then measuring the fluorescence on the Luminex system. These experiments, like the one showed in FIG. 3 determined that an incubation of 40 minutes is optimum for the sensitivity of the test and increased the fluorescence signal by about 2 fold.
Example 3
Typing of HPV
Specificity and sensitivity for each type was determined by adding PCR product from a known source of HPV, clones carrying the whole HPV genome, when available, or clones of the MY region of the genome amplified by PCR form clinical samples or synthesized using published genomic sequences (see Material and methods for a complete list). All clones were confirmed by direct sequencing and comparison with published HPV sequences.
Using the PCR amplification method, exonuclease digestion and microsphere hybridization described above, amplified HPV DNA from each type was hybridized to a mixture of the 46 types of microspheres carrying the 46 specific HPV probes. After hybridization, the microsphere mixture was analyzed by the Luminex LiquidChip 200 flow cytometer. Four negative controls, containing only host cell DNA, were run alongside the samples. The average background fluorescence of each bead in the controls was subtracted from the fluorescence of each bead of the samples. This type of background correction is necessary because different bead types may have different background fluorescence. This corrections avoids the need for a bead washing step, used in other Luminex procedures (Wallace et al., 2005; Oh et al., 2007; Schmitt et al., 2006; Jiang et al., 2006; Seme et al., 2009). A fluorescence signal greater than 100 FU after correction was chosen as threshold for positivity.
The complete results of are shown in Table 4, where each column represents the fluorescence associated with the microsphere carrying the probe for the indicated HPV type in the presence of the HPV DNA of the types indicated on the leftmost column. FIG. 4 shows the same results in graphic format. It can be seen that all the 46 probes strongly hybridize with the corresponding HPV DNA, but not with HPV DNA of different types. It should be noted that in the particular experiment shown in Table 4 and FIG. 4, the microsphere for HPV 89 also shows fluorescence above the 100 FU threshold level in the presence of HPV 44 DNA (513 FU), the microsphere for HPV 72 in the presence HPV 81 DNA (118 FU) and the microsphere for HPV 44 in the presence of HPV 86 DNA (391 FU). This should be interpreted as random fluctuations, rather than systematic cross-reactivity, because the abnormal fluorescence reading was not reproducible in other experiments. This corresponds to a false positive rate of 3/1980 measurements or 0.15%. To avoid false positives, clinical samples are tested in duplicate and the measurement is repeated if the duplicates give discordant results.
We then tested the ability of the Luminex method to detect infections with multiple types in the same sample, by amplifying DNAs from different HPV types separately and then mixing them together in a single Luminex detection reaction. The amount of DNA per type was kept constant, to simulate the situation of clinical samples, in which a mixture of different DNA is amplified to the maximum capacity of the PCR reaction, regardless of the number of types present. The results are presented in FIG. 5, that shows that at least 30 different types can be detected simultaneously with minimal cross hybridization. Some false negatives and false positives are however present. The false negatives are probably due to the fact that the fluorescence for each HPV type is low when many types are present and therefore some microsphere may fall under the 50 FU that was established as positivity threshold. False positive for HPV 72 are due to fluctuation in the background fluorescence of this microsphere.
Example 4
Validation Using Clinical Samples—Direct Sequencing
Validation against clinical samples was performed by comparing the results of the NML Luminex genotyping method with direct sequencing of the amplified products. Because direct sequencing identifies any HPV type without misclassification, this is a further test of the specificity of the probes of the NML Luminex assay.
Seven hundred seventy five samples were amplified by nested PCR as described above and the products were typed with the NML Luminex method. The same samples were amplified separately by nested PCR and run on an agarose gel to determine the presence of HPV DNA. Positive samples were sent for sequencing at the NML DNA Core facility, using GP5+ and GP6+ primers to sequence both strands of the amplified products. The assembled sequenced was compared against GenBank sequences using BLAST (Altschul et al., 1990). Type identification required a nucleotide identity greater that 90% on a fragment of at least 60 nucleotide in length.
The results presented in Table 5, show that the two methods were 97.7% concordant for the detection of HPV, regardless of type. The sensitivity and specificity of the NML Luminex method vs direct sequencing, taken as a gold standard, were 98.8% (97.1-99.6, 95% CI) and 96.4% (96.4-93.8, 95% CI), respectively.
When positive identification of HPV type is taken into consideration, the direct sequencing method could not determine the sequence of 34 positive samples, 32 of which were typed by the NML Luminex method. There was no agreement on the HPV type detected for 13 out of 429 samples positive with both methods (3.3%). The NML Luminex method detected a total of 793 HPV types, vs 577 for direct sequencing. This discrepancy is due to the fact that direct sequencing cannot detect multiple HPV types present in the same sample.
A breakdown of HPV types detected by the two methods is presented in Table 6.
From the validation against the direct sequencing method, it is impossible to establish if the extra types detected by the NML Luminex assay are due to better sensitivity for multiple infections or to poor specificity.
Example 5
Validation Using Clinical Samples—Comparison to Roche Linear Assay
Therefore we compared the performance of the NML Luminex assay using the Roche LinearArray HPV genotyping method as the gold standard. The Linear Array kit can detect 37 different genotypes and its amplification system, based on the PGMY primers, is particularly efficient in amplifying multiple types. Linear Array is FDA approved and it is one of the standard methods used in the literature on HPV epidemiology.
For this comparison we used a set of 880 samples that were tested for HPV at the National Microbiology Laboratory in parallel by the Roche Linear Array kit, according to the instruction of the manufacturer, and by the NML Luminex genotyping method.
The Linear Array probe for HPV 52 cross-reacts with HPV type 33, 35 and 58. Therefore all Linear Array HPV 52 results were confirmed by real time PCR specific for HPV 52 as previously described (Coutlee et al., 2007). Linear Array contains a probe designated HPV 55, but according to the latest classification of HPVs (de Villier et al, 2004) the type 55 is considered a variant of HPV 44 and HPV 55 has been removed. Therefore in this work we used the HPV 44 designation. Roche Linear Array probes IS39 and CP6108 recognize types more recently designated as HPV 82 and HPV 89 respectively. The more recent designation was used in this work.
Table 7 shows a comparison of the NML Luminex with Roche linear array for the detection of positive samples for any HPV type. Discordant results are 7% overall and 5.2% of discordant samples tested positive with the NML Luminex but not with the linear array. This is due to the greater sensitivity of the nested PCR used for the NML Luminex method and to the detection of HPV types not present on the linear array set of probes.
Table 8 shows the comparison of the NML Luminex method with the Roche Linear array for the detection of all HPV types and multiple infections. The Roche Linear array detected considerably more types of HPV (1111 vs. 917), due to the better performance in samples with high numbers of multiple infections. This reduced performance for multiple infections is not due is not a problem with the Luminex detection system, which can detect at least 32 different types simultaneously, as shown above (FIG. 5), but it is a shortcoming of the PCR amplification step, which is less efficient when a mixture of different types is present. Table 9 shows the comparison results for the individual types. Apart from the types not detected by the Roche Linear Array (HPV 13, 32, 74, 85, 86, 87, 90 and 91) the detection of HPV types 52, 53, 61, 73 84 and 89 was statistically significantly more sensitive (X2 test) in the Roche linear Array, while the detection of HPV type 67 was more sensitive in the NML Luminex.
Table 10 shows the results after exclusion of samples with multiple infections with 4 or more types, as determined by the Roche linear array. This Table shows a much better concordance between NML Luminex and Roche Linear Array with respect of total number of types detected (535 vs 534, respectively) and type breakdown. In addition to the types not detected by the Roche Linear Array, only type 52 (better detection for Linear Array) and type 67 (better detection for NM Luminex) are now significantly different.
While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.
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TABLE 1







Sequences













SEQ



Sequence

ID



Definition
Probe Sequence
NO:















HPV 6
ACCACACGCAGTACCAACAT
47



 



HPV 6
CATGCGTCATGTGGAAGAGT
48



 



HPV11
ATGCGCCATGTGGAGGAGTT
49



 



HPV11
TGGTAGATACCACACGCAGT
50



 



HPV13
TGACTGTGTGTGCAGCCACT
51



 



HPV13
GTTGATACTACACGCAGTAC
52



 



HPV16
ACCTACGACATGGGGAGGAA
53



 



HPV16
ATGTCATTATGTGCTGCCAT
54



 



HPV18
CAGTCTCCTGTACCTGGGCA
55



 



HPV18
AGATACCACTCCCAGTACCA
56



 



HPV26
CCTGTGTTGATACCACCCGC
57



 



HPV26
CAGCATCTGCATCCACTCCA
58



 



HPV30
TGGACACCACTAGGAACACA
59



 



HPV30
ATCTGCAACCACACAAACGT
60



 



HPV31
TGTCTGTTTGTGCTGCAATT
61



 



HPV31
AGATACCACACGTAGTACCA
62



 



HPV32
ATCTACGCCATGCAGAGGAA
63



 



HPV32
ACTGTTGTGGATACTACCCG
64



 



HPV33
TGGTAGATACCACACGCAGT
65



 



HPV33
GCACACAAGTAACTAGTGAC
66



 



HPV34
CCACAAGTACAACTGCACCA
67



 



HPV34
ACCTCAGACATGCAGAAGAG
68



 



HPV35
TGTCTGTGTGTTCTGCTGTG
69



 



HPV35
AGGCATGGTGAAGAATATGA
70



 



HPV39
ACTGTTGTGGACACTACCCG
71



 



HPV39
TACCAGGCACGTGGAGGAGT
72



 



HPV40
ATGTGCTGCCACACAGTCCC
73



 



HPV40
TTTGCGTCATGGGGAGGAGT
74



 



HPV42
GCCACTGCAACATCTGGTGA
75



 



HPV42
ACTGTGGTTGATACTACCCG
76



 



HPV44
GTGCTGCCACTACACAGTCC
77



 



HPV44
CATGCGACATGTTGAGGAGT
78



 



HPV45
GTGGACACTACCCGCAGTAC
79



 



HPV45
GTGCCAAGTACATATGACCC
80



 



HPV47
TTACTCTCAGGCAGGGGACA
81



 



HPV47
GTCACAGTTGTAGACAACAC
82



 



HPV51
GCACTGCCACTGCTGCGGTT
83



 



HPV51
AGGCATGGGGAAGAGTATGA
84



 



HPV52
ACCTTCGTCATGGCGAGGAA
85



 



HPV52
TGGATACCACTCGTAGCACT
86



 



HPV53
ACTCTTTCCGCAACCACACA
87



 



HPV53
TGTTGTGGATACCACCAGGA
88



 



HPV54
GCTACAGCATCCACGCAGGA
89



 



HPV54
CAGTTGTAGATACCACCCGT
90



 



HPV56
ACCTTAGACATGTGGAGGAA
91



 



HPV56
CTGCTACAGAACAGTTAAGT
92



 



HPV58
GGTTGATACCACTCGTAGCA
93



 



HPV58
TGCACTGAAGTAACTAAGGA
94



 



HPV59
ACTACTCGCAGCACCAATCT
95



 



HPV59
ATGCCAGACATGTGGAGGAA
96



 



HPV61
CCGTTGTGGATACCACCCGC
97



 



HPV61
TTGCGCCATACAGAGGAGTT
98



 



HPV62
TGTACCGCCTCCACTGCTGC
99



 



HPV62
TTTGCGACACACGGAGGAAT
100



 



HPV66
ACACACGCCATGTAGAGGAA
101



 



HPV66
ACCAGAAGCACCAACATGAC
102



 



HPVG7
ACACGTAGTACCAACATGAC
103



 



HPV67
ACCTTAGACATGTGGAAGAA
104



 



HPV68
TTGTGGATACAACGCGCAGT
105



 



HPV68
CAGACTCTACTGTACCAGCT
106



 



HPV69
ACCCGCAGTACCAACCTCAC 
107



 



HPVG9
GCACAATCTGCATCTGCCAC
108



 



HPV70
TCTGCCTGCACCGAAACGGC
109



 



HPV70
ACTGTGGTGGACACTACACG
110



 



HPV71
ATGTCCATCTGTGCTACCAA
111



 



HPV71
ACAGTTGTGACACATCACGT
112



 



HPV72
ACTGCCACAGCGTCCTCTGT
113



 



HPV72
ATCTTCGCCACACTGAGGAA
114



 



HPV73
GGTACACAGGCTAGTAGCTC
115



 



HPV73
CTACAACGTATGCCAACTCT
116



 



HPV74
ACCTCACAATCGCCTTCTGC
117



 



HPV74
TGGATACCACACGCAGTACT
118



 



HPV82
GCACTGCTGTTACTCCATCT
119



 



HPV82
AGCAGTACATTAGGCATGGG
120



 



HPV82
GCACTGCTGCTACTCCATCA
121



 



HPV82
GCACAGACATTCACTCCAAC
122



 



HPV83
GCTGCTGCTACACAGGCTAA
123



 



HPV83
ACCTCCGCCACACAGAGGAA
124



 



HPV84
AGATACCACCCGCAGCACCA
125



 



HPV84
AGTGCTGCTACCAACACCGA
126



 



HPV85
ACACACGCCATGTAGAGGAA
127



 



HPV85
ACTGTGGTAGACACAACACG
128



 



HPV85
AGTGCCGCTACCCAGAAGGC
129



 



HPV86
TCGACACCACCCGCAGTACT
130



 



HPV87
TGCTGCCACTCAAACAACCA
131



 



HPV87
CGGTTGTTGATACTACTCGC
132



 



HPV89
GTGCTGCTTCCCAGTCTGGC
133



 



HPV89
ACCACCCGTAGTACCAACCT
134



 



HPV91
TGTGGATACAACTCGCAGCA
135



 



HPV91
GCATCCACTGAGTCTGTGCT
136

















TABLE 2







History of Probe Development












HPV


BEAD

SEQ


TYPE
OLIGO SEQUENCE 5′ TO 3′
DNA SOURCE
#
COMMENTS
ID NO:















6
ACTACACGCAGTACCAACATGACATTATGT
PLASMID
50
low hybridization
137



CGTAACTACATCTTCCACATACACCAATTC
PLASMID
50
no hybridization
138



CAACATGACATTATGTGCATCCGTAACTAC
PLASMID
50
no hybridization
139



CATGACATTATGTGCATCCGTAACTACATCTTC
PLASMID
50
no hybridization
140



CATCTTCCACATACACCAATTCTGATTATA
PLASMID
50
ok
1



TCCGTAACTACATCTTCCACATACACCAAT
PLASMID
50
no hybridization
141


 


11
ACTATGTGCATCTGTGTCTAAATCTGCTAC
PLASMID
13
good
2


 


13
AGCCACTACATCATCTCTTTCAGACACATA
PLASMID
34
no hybridization
142



TAACATGACTGTGTGTGCAGCCACTACATC
PLASMID
34
no hybridization
143



GTGTGTGCAGCCACTACATCATCTCTTTCA
PLASMID
34
no hybridization
3



GTGTGTGCAGCCACTACATCATCTCTTTCA
PLASMID
34
good
3


 


16
GCCATATCTACTTCAGAAACTACATATAAA
PLASMID
98
no hybridization
144



AAATATGTCATTATGTGCTGCCATATCTAC
PLASMID
98
no hybridization
4



ATGTCATTATGTGCTGCCATATCTACTTCA
PLASMID
98
cross hybridization 
145






with 62




GTCATTATGTGCTGCCATATCTACTTCAGA
PLASMID
98
cross hybridization
146



GCCATATCTACTTCAGAAACTACATATAAA
PLASMID
98
no hybridization
147


 


18
ATATGTGCTTCTACACAGTCTCCTGTACCT
PLASMID
15
good
5



AACAATATGTGCTTCTACACAGTCTCCTGT
PLASMID
15
cross hybridization
148



TCCTGTACCTGGGCAATATGATGCTACCAA
PLASMID
15
cross hybridization
149



TATGTGCTTCTACACAGTCTCCTGTACCTG
PLASMID
15
no hybridization
150



TATGTGCTTCTACACAGTCTCCTGTACCTG
PLASMID
15
no hybridization
150


 


26
CCTTACCATTAGTACATTATCTGCAGCATC
PLASMID
5
good
6



AACCTTACCATTAGTACATTATCTGCAGCA
PLASMID
5
no hybridization
151



ACATTATCTGCAGCATCTGCATCCACTCCA
PLASMID
5
good
152


 


30
ATCTGCAACCACACAAACGTTATCCACATA
PLASMID
53
good
7



CCACACAAACGTTATCCACATATAATTCAA
PLASMID
40
no hybridization
153



GACTATATCTGCAACCACACAAACGTTATC
PLASMID
40
no hybridization
154



ATCTGCAACCACACAAACGTTATCCACATA
PLASMID
40
no hybridization
155


 


31
AAGTAGTAATTTTAAAGAGTATTTAAGACA
PLASMID
55
no hybridization
156



ATGTCTGTTTGTGCTGCAATTGCAAACAGT
PLASMID
55
no hybridization
157



CAATATGTCTGTTTGTGCTGCAATTGCAAA
PLASMID
55
low hybridization
8



CAATATGTCTGTTTGTGCTGCAATTGCAAA
PLASMID
25
no hybridization
8



CAATATGTCTGTTTGTGCTGCAATTGCAAA
PLASMID
25
no hybridization
8



AACAGTGATACTACATTTAAAAGTAGTAAT
PLASMID
25
no hybridization
158



TCTGTTTGTGCTGCAATTGCAAACAGTGAT
PLASMID
25
no hybridization
159



GCAATTGCAAACAGTGATACTACATTTAAA
PLASMID
55
no hybridization
160


 


32
ACTGTAACAACTGAAGACACATACAAGTCT
Test Sample 4330
36
good
9


7
CACCAACACCATATGACAATAGTAAGTTTA
Test Sample 3228
33
take out not mucosal
161


 


33
TAGTGACAGTACATATAAAAATGAAAATTT
PLASMID
58
cross hybridization
162



TAATATGACTTTATGCACACAAGTAACTAG
PLASMID
30
ok
10



GCACACAAGTAACTAGTGACAGTACATATA
PLASMID
58
no hybridization
163



GTGACAGTACATATAAAAATGAAAATTTTA
PLASMID
30
ok
164


34
TAGGTACACAATCCACAAGTACAACTGCAC
PLASMID
37
no dna sample
165


 


35
TGTCTGTGTGTTCTGCTGTGTCTTCTAGTG
Test Sample 4498
30
no hybridization
166



GTGTCTTCTAGTGACAGTACATATAAAAAT
Test Sample 4498
30
no hybridization
167



AACCCGTAGTACAAATATGTCTGTGTGTTC
Test Sample 4498
77
no hybridization
168



AAATATGTCTGTGTGTTCTGCTGTGTCTTC
Test Sample 4498
77
good
11



TCTGCTGTGTCTTCTAGTGACAGTACAATA
Test Sample 4498
77
no hybridization
169


 


39
CTTTACATTATCTACCTCTATAGAGTCTTC
Test Sample 4317
25
low hybridization
170



AGAGTCTTCCATACCTTCTACATATGATCC
Test Sample 4317
25
low hybridization
171



CCGTAGTACCAACTTTACATTATCTACCTC
PLASMID
76
cross hybridization
172



CAACTTTACATTATCTACCTCTATAGAGTC
PLASMID
76
no hybridization
173



ATCTACCTCTATAGAGTCTTCCATACCTTC
PLASMID
76
good
12



CTACCTCTATAGAGTCTTCCATACCTTTCT
Test Sample 4317
76
dna sample no good
174


 


40
GTCCCCCACACCAACCCCATATAATAACAG
TEST SAMPLE 3343
38
cross hybridization
175



CTTATGTGCTGCCACACAGTCCCCCACACC
TEST SAMPLE 3343
38
ok
13



ACCCCATATAATAACAGTAATTTCAAGGAA
TEST SAMPLE 3343
38
cross hybridization
176



ACAGTCCCCCACACCAACCCCATATAATAA
TEST SAMPLE 3343
38
cross hybridization
177


 


42
TCTGGTGATACATATACAGCTGCTAATTTT
test sample
42
multiple infection
14



ACATCTGGTGATACATATACAGCTGCTAAT
none
42
cross hybridization
178



CACTGCAACATCTGGTGATACATATACAGC
Test Sample 3398
42
dna sample not good
179


 


43
AAACTTAACGTTATGTGCCTCTACTGACCC
none
64
waiting
15



TGACCCTACTGTGCCCAGTACATATGACAA
none
64
cross hybridization
180



TGCAAAGTTTAAGGAATACCTGCGGCA
none
64
cross hybridization
181


 


44
GCCACTACACAGTCCCCTCCGTCTACATAT
PLASMID
3
no hybridization
182



GACAATATGTGCTGCCACTACACAGTCCCC
PLASMID
10
ok
183



AAACATGACAATATGTGCTGCCACTACACA
PLASMID
10
good
16



AATATGTGCTGCCACTACACAGTCCCCTCC
PLASMID
10
cross hybridization
184


 


45
TAATTTAACATTATGTGCCTCTACACAAAA
PLASMID
18
good
17



TAACATTATGTGCCTCTACACAAAATCCTG
Plasmid
18
no hybridization
185



CTCTACACAAAATCCTGTGCCAAGTACATA
PLASMID
18
low hybridization
186


 


51
TTAACTATTAGCACTGCCACTGCTGCGGTT
Test Sample 98066
28
no hybridization
187



GCCACTGCTGCGGTTTCCCCACATTTACTC
Test Sample 98066
28
no hybridization
18



TTTAACTATTAGCACTGCCACTGCTGCGGT
Test Sample 98066
28
no hybridization
188



TAGCACTGCCACTGCTGCGGTTTCCCCAAC
Test Sample 98066
28
no hybridization
189


 


52
CACAGTTGTGGATACCACTCGTAGCACTAA
PLASMID
23
no hybridization
190



AAAAGGAAAGCACATATAAAAATGAAAATT
PLASMID
23
no hybridization
191



CACTGCTAGCACTAACATGACTTTATGTGC
PLASMID
8
no hybridization
192



CATGACTTTATGTGCTGAGGTTAAAAAGGA
PLASMID
8
no hybridization
193



AGCACATATAAAAATGAAAATTTTAAGGAA
PLASMID
8
no hybridization
194



GACTTTATGTGCTGAGGTTAAAAAGGAAAG
PLASMID
8
no hybridization
19



AAAAGGAAAGCACATATAAAAATGAAAATT
PLASMID
8
no hybridization
195



GAGGTTAAAAAGGAAAGCACATATAAAAAT
PLASMID
8
cross hybridization
196


 


53
GATCTCTTTCCGCAACCACACAGTCTATGT
PLASMID
44
no hybridization
197


53
CTCTTTCCGCAACCACACAGTCTATGTCTA
PLASMID
44
no hybridization
198


53
CTACATATAATTCAAAGCAAATTAAACAGT
PLASMID
44
no hybridization
199


53
CGCAACCACACAGTCTATGTCTACATATAA
PLASMID
44
good
20


54
ACAGCATCCACGCAGGATAGCTTTAATAAT
Plasmid
65
good
21


56
AGTTAAGTAAATATGATGCACGAAAAATTA
PLASMID
35
cross hybridization
200


56
GTACTGCTACAGAACAGTTAAGTAAATATG
PLASMID
35
no hybridization
201


56
TAGAAGTACTAACATGACTATTAGTACTGC
PLASMID
35
no hybridization
202


56
CATGACTATTAGTACTGCTACCAGAACAGT
PLASMID
35
good
22


58
TGCACTGAAGTAACTAAGGAAGGTACATAT
Test Sample 98028
43
no hybridization
203


58
CACTAATATGACATTATGCACTGAAGTAAC
Test Sample 98028
43
some cross hybridization
204


58
ATGACATTATGcAcTGAAGTAAcTAAGGAA
Test Sample 98028
43
good
23


58
ACTAAGGAAGGTACATATAAAAATGATAAT
Test Sample 98028
43
no hybridization
205


59
ctttctgtgtgtgcttctactacttcttct
PLASMID
60
good
24






significant amounts of
206


59
ACTACTCGCAGCACCAATCTTTCTGTGTGT
PLASMID
60
cross hybridization



61
CATTTGTACTGCTACATCCCCCCCTGTATC
PLASMID

good
25


61
TAATTTAACCATTTGTACTGCTACATCCCC
Plasmid
46
no hybridization
207


61
AACCATTTGTACTGCTACATCCCCCCCTGT
PLASMID
46
low hybridization
208


62
ACCGCCTCCACTGCTGCAGCAGAATACACG
Test sample N34
66
good
26


66
TGAAATCAATCAATACCTTCGCCATGTGGA
Test Sample 99081
40
no hybridization
209


66
GACTATTAATGCAGCTAAAAGCACATTAAC
Test sample 4491
75
good
27


66
AGCTAAAAGCACATTAACTAAATATGATGC
Test sample 4491
75
no hybridization
210


66
TAATGCAGCTAAAAGCACATTAACTAATAT
Test sample 4491
75
cross hybridization
211


66
TTAACTAAATATGATGCCCGTGAAATCAAT
SAMPLE 4491
75
cross hybridization
212


66
TAATGCAGCTAAAAGCACATTAACTACATA
Test sample 4491
20
cross hybridization
213


67
AAAAATCAGAGGCTACATACAAAAATGAAA
PLASMID
200
waiting
214


67
TCTGAGGAAAAATCAGAGGCTACATACAAA
PLASMID
200
good
28


68
ATTGTCCACTACTACAGACTCTACTGTACC
none
45
no dna sample
29


69
ACTGTATCTGCACAATCTGCATCTGCCACT
none
72
no dna sample
30


70
GTCTGCCTGCACCGAAACGGCCATACCTGC
Test Sample 4190
47
good
31


71
ACCAAAACTGTTGAGTCTACATATAAAGCC
none
73
no dna sample
32


72
CAGCTTCTAATTTTCGTGAGTATCTTCGCC
PLASMID
51
good
33


72
CACAGCGTCCTCTGTATCAGAATATTACAG
PLASMID
51
good
215


73
TAGGTACACAGGCTAGTAGCTCTACTACAA
PLASMID
52
good
34


74
TAACATGACTGTGTGTGCTCCTACCTCACA
Plasmid
54
good
35


74
CTCACAATCGCCTTCTGCTACATATAATAG
PLASMID
54
no hybridization
216


81
CACAGCTACATCTGCTGCTGCAGAATACAA
PLASMID
56
cross hybridization
217


81
TACTATTTGCACAGCTACATCTGCTGCTGC
Plasmid
56
good
36


81
ATCTGCTGCTGCAGAATACAAGGCCTCTAA
PLASMID
56
low hybridization
218


82
GCTGTTACTCCATCTGTTGCACAAACATTT
PLASMID
61
good
37


83
CAGCTGCTGCTACACAGGCTAATGAATACA
test sample 22038
74
no dna sample
38


84
GCTACCAACACCGAATCAGAATATAAACCT
test sample 21A
17
no dna sample
39


85
TGCAACTACTAATCCAGTTCCATCTATATA
none
19
no dna sample
40


86
CCCCTCTAAGTTTAATGAATATCTAAG
none
20
cross hybridization
219


86
TAATTTTACTATTAGTGCCGCTACCCAGAA
none
20
no dna sample
41


86
TCTGAATATGACCCCCTCTAAGTTTAATGA
none
20
cross hybridization
220


86
CGCTACCCAGAAGGCCTCTGAATATGACCC
none
20
cross hybridization
221


87
TGCCACTCAAACAACCACTGAATATGACCC
none
62
cross hybridization
222


87
CAATTTTACTATTAGTGCTGCCACTCAAAC
none
62
no dna sample
42


87
CACAAAGTTTAAGGAATATTTAAGGCA
none
62
cross hybridization
223


87
AACAACCACTGAATATGACCCCACAAAGTT
none
62
cross hybridization
224


89
GCTTCCCAGTCTGGCACAGAATAC
none
23
good
43


89
CCGTAGTACCAACCTTACCATTTGTGCTGC
none
23
cross hybridization
225


89
CATTTGTGCTGCTTCCCAGTCTGGCACAGA
none
23
cross hybridization
226


90
CACCAATATGACTATTTGTGCCACACAAAC
test sample 4015
83
no dna sample
44


90
CACACAAACACCCTCTGACACATACAAGGC
test sample 4015
83
cross hybridization
227


91
TAACTTAACCTTGTGTGCATCCACTGAGTC
test sample 50211 
63
hybridizes with 89
45


91
CTACCTACTACATATGACAACACAAAGTTC
find new dna
63
no hybridization 
228


91
ATCCACTGAGTCTGTGCTACCTACTACATA
find new dna
63
no hybridization 
229


97
TTTAACACTGTGTGCTTCTACACAAAATGG
PLASMID

Fair
230


97
TCTACACAAAATGGCGTAGCTACCACATAT
PLASMID

good
46
















TABLE 3







Probe Sequences


Final list of probes for the detection


 of 46 HPV types with the Luminex 


  Microsphere technology.









SEQ ID
HPV



NO:
TYPE
OLIGO SEQUENCE 5′ TO 3′












1
6
CATCTTCCACATACACCAATTCTGATTATA


 


2
11
ACTATGTGCATCTGTGTCTAAATCTGCTAC


 


3
13
GTGTGTGCAGCCACTACATCATCTCTTTCA


 


4
16
AAATATGTCATTATGTGCTGCCATATCTAC


 


5
18
ATATGTGCTTCTACACAGTCTCCTGTACCT


 


6
26
CCTTACCATTAGTACATTATCTGCAGCATC


 


7
30
ATCTGCAACCACACAAACGTTATCCACATA


 


8
31
CAATATGTCTGTTTGTGCTGCAATTGCAAA


 


9
32
ACTGTAACAACTGAAGACACATACAAGTCT


 


10
33
TAATATGACTTTATGCACACAAGTAACTAG


 


11
35
AAATATGTCTGTGTGTTCTGCTGTGTCTTC


 


12
39
ATCTACCTCTATAGAGTCTTCCATACCTTC


 


13
40
CTTATGTGCTGCCACACAGTCCCCCACACC


 


14
42
TCTGGTGATACATATACAGCTGCTAATTTT


 


15
43
AAACTTAACGTTATGTGCCTCTACTGACCC


 


16
44
AAACATGACAATATGTGCTGCCACTACACA


 


17
45
TAATTTAACATTATGTGCCTCTACACAAAA


 


18
51
GCCACTGCTGCGGTTTCCCCACATTTACTC


 


19
52
GACTTTATGTGCTGAGGTTAAAAAGGAAAG


 


20
53
CGCAACCACACAGTCTATGTCTACATATAA


 


21
54
ACAGCATCCACGCAGGATAGCTTTAATAAT


 


22
56
CATGACTATTAGTACTGCTACCAGAACAGT


 


23
58
ATGACATTATGCACTGAAGTAACTAAGGAA


 


24
59
CTTTCTGTGTGTGCTTCTACTACTTCTTCT


 


25
61
CATTTGTACTGCTACATCCCCCCCTGTATC


 


26
62
ACCGCCTCCACTGCTGCAGCAGAATACACG


 


27
66
GACTATTAATGCAGCTAAAAGCACATTAAC


 


28
67
TCTGAGGAAAAATCAGAGGCTACATACAAA


 


29
68
ATTGTCCACTACTACAGACTCTACTGTACC


 


30
69
ACTGTATCTGCACAATCTGCATCTGCCACT


 


31
70
GTCTGCCTGCACCGAAACGGCCATACCTGC


 


32
71
ACCAAAACTGTTGAGTCTACATATAAAGCC


 


33
72
CAGCTTCTAATTTTCGTGAGTATCTTCGCC


 


34
73
TAGGTACACAGGCTAGTAGCTCTACTACAA


 


35
74
TAACATGACTGTGTGTGCTCCTACCTCACA


 


36
81
TACTATTTGCACAGCTACATCTGCTGCTGC


 


37
82
GCTGTTACTCCATCTGTTGCACAAACATTT


 


38
83
CAGCTGCTGCTACACAGGCTAATGAATACA


 


39
84
GCTACCAACACCGAATCAGAATATAAACCT


 


40
85
TGCAACTACTAATCCAGTTCCATCTATATA


 


41
86
TAATTTTACTATTAGTGCCGCTACCCAGAA


 


42
87
CAATTTTACTATTAGTGCTGCCACTCAAAC


 


43
89
GCTTCCCAGTCTGGCACAGAATAC


 


44
90
CACCAATATGACTATTTGTGCCACACAAAC


 


45
91
TAACTTAACCTTGTGTGCATCCACTGAGTC


 


46
97
TCTACACAAAATGGCGTAGCTACCACATAT
















TABLE 4





Determination of specificity and sensitivity of the 46  


probes (columns) for hybridization with the


  cognate HPV DNA amplified from cloned


controls for each HPV type (rows).






















Sample
HPV 6
HPV 11
HPV 13
HPV 16
HPV 18
HPV 26
HPV 30





HPV 6
2004
0
2
0
0
0
0


HPV 11
17
1445
−8
−7
57
5
−5


HPV 13
1
3
1061
1
−17
−12
8


HPV 16
0
0
0
3378
0
0
0


HPV 18
0
0
0
15
2802
0
0


HPV 26
0
5
0
0
0
1961
0


HPV 30
0
0
0
0
0
0
5008


HPV 31
0
0
0
0
0
0
0


HPV 32
0
0
0
0
0
0
0


HPV 33
3
0
0
6
0
0
0


HPV 35
0
0
0
0
0
0
0


HPV 39
0
0
0
0
0
0
0


HPV 40
0
0
0
0
0
0
0


HPV 42
0
0
0
0
0
0
0


HPV 43
0
0
0
0
0
0
0


HPV 44
-9
4
7
−16
15
5
2


HPV 45
0
0
0
0
0
0
0


HPV 51
0
0
0
0
0
0
0


HPV 52
5
14
3
−5
16
−4
−13


HPV 53
0
0
0
0
0
0
0


HPV 54
0
0
0
0
0
0
0





Sample
HPV 6
HPV 11
HPV 13
HPV 16
HPV 18
HPV 26
HPV 30





HPV 54
0
0
0
0
0
0
0


HPV 56
0
0
0
0
0
0
0


HPV 58
0
0
0
0
0
0
0


HPV 59
0
0
0
0
0
0
0


HPV 61
0
0
0
0
0
0
0


HPV 62
−4
3
3
4
−16
−13
−17


HPV 66
0
0
0
0
0
0
0


HPV 67
0
0
0
0
0
0
0


Hpv 68
8
25
−7
1
−2
−15
23


HPV 69

8
−3
1
−15
7
−13


HPV 70
0
0
0
0
0
0
0


HPV 71

2
−9
17
9
6
−77


HPV 72

0
0
0
0
0
0


HPV 73

5
34
15
2
12
18


HPV 74

5
7
−1
3
−11
18


HPV 81

14
−5
−18
−18
−6
7


HPV 82
0
0
0
0
0
0
0


HPV 83

0
−11
−4
1
−19
−6


HPV 84

−10
−9
−6
10
−2
−2


HPV 85
0
0
0
0
0
0
0


HPV 86

7
6
13
32
−1
7


HPV 87

−9
5
−18
−6
13
−13


HPV 89

−5
10
5
4
3
−4


HPV 90

15
−11
9
13
2
−2


HPV 91

19
10
22
14
−2
1


HPV 97

−8
−5
−3
−1
12
7

















Sample
HPV 31
HPV 32
HPV 33
HPV 35
HPV 39
HPV 40
HPV 42





HPV 6
0
0
5
0
0
0
0


HPV 11
−23
1
−91
10
0
−13
−3


HPV 13
−45
−2
−106
−13
−6
13
−11


HPV 16
0
9
9
7
0
0
0


HPV 18
1
0
0
0
0
0
0


HPV 26
0
9
0
0
2
0
0


HPV 30
0
0
17
0
0
0
0


HPV 31
1168
0
27
0
11
0
0


HPV 32
0
2601
5
0
0
0
0


HPV 33
0
2
1083
0
0
0
2


HPV 35
0
0
13
2348
1
0
0


HPV 39
0
0
0
0
3554
0
0


HPV 40
0
0
0
0
0
4154
0


HPV 42
0
0
0
0
0
0
3689


HPV 43
0
0
0
0
0
0
0


HPV 44
−23
−4
12
0
−3
1
−7


HPV 45
0
0
0
0
0
0
0


HPV 51
0
0
0
2
0
0
0


HPV 52
4
11
−24
23
2
13
−13


HPV 53
0
0
0
0
0
0
0


HPV 54
0
0
0
0
0
0
0

















Sample
HPV 31
HPV 32
HPV 33
HPV 35
HPV 39
HPV 40
HPV 42





HPV 54
0
0
0
0
0
0
0


HPV 56
0
0
0
0
0
0
0


HPV 58
0
0
0
0
0
0
0


HPV 59
0
0
0
0
0
0
0


HPV 61
0
0
0
0
0
0
0


HPV 62
−10
−41
−6
7
0
−3
−4


HPV 66
0
0
0
0
0
0
0


HPV 67
0
0
0
0
0
0
0


Hpv 68
4
1
−62
−5
3
−11
3


HPV 69
−34
25
−56
0
4
3
2


HPV 70
0
0
0
0
0
0
0


HPV 71
−7
−58
−7
−9
13
12
−8


HPV 72
0
0
0
14
0
0
0


HPV 73
32
12
23
22
18
2
22


HPV 74
−4
−1
−40
−4
4
6
20


HPV 81
30
9
59
18
−3
1
34


HPV 82
0
0
0
0
0
0
0


HPV 83
−7
9
−46
7
−17
−3
14


HPV 84
−12
7
−4
9
18
7
−4


HPV 85
0
0
0
0
0
0
0


HPV 86
−15
23
−10
16
−3
1
12


HPV 87
−10
−7
−46
−13
7
3
6


HPV 89
−18
15
−5
−13
−5
2
−2


HPV 90
8
10
−6
33
0
32
43


HPV 91
17
3
8
−3
22
13
9


HPV 97
−2
4
−30
4
−4
−7
−2

















Sample
HPV 43
HPV 44
HPV 45
HPV 51
HPV 52
HPV 53
HPV 54





HPV 6
0
0
0
0
0
0
0


HPV 11
−15
−9
−19
15
−5
−7
−6


HPV 13
−6
−12
−14
−30
5
8
−11


HPV 16
0
5
0
0
0
0
0


HPV 18
0
8
5
0
0
0
0


HPV 26
9
0
0
18
0
0
0


HPV 30
0
6
14
0
0
0
6


HPV 31
0
0
0
0
0
0
0


HPV 32
0
0
0
0
0
0
0


HPV 33
4
0
0
8
0
0
0


HPV 35
0
3
0
0
0
0
0


HPV 39
0
0
0
0
0
0
0


HPV 40
0
0
0
0
0
0
0


HPV 42
0
0
0
0
0
0
0


HPV 43
3364
0
0
0
0
0
0


HPV 44
27
2068
−8
−31
0
14
2


HPV 45
0
0
1922
0
0
0
0


HPV 51
0
0
0
2884
0
0
0


HPV 52
0
6
22
−20
2429
−10
−5


HPV 53
0
0
0
0
0
2162
0


HPV 54
0
0
0
0
0
0
2755

















Sample
HPV 43
HPV 44
HPV 45
HPV 51
HPV 52
HPV 53
HPV 54





HPV 54
0
0
0
0
0
0
2755


HPV 56
0
0
0
0
0
0
0


HPV 58
0
0
0
0
0
0
0


HPV 59
0
0
0
0
0
0
0


HPV 61
0
0
0
0
0
0
0


HPV 62
12
0
−6
−5
1
−3
−19


HPV 66
0
0
0
0
0
0
0


HPV 67
0
0
0
0
0
0
0


Hpv 68
8
−12
−3
−14
−7
8
6


HPV 69
8
9
3
−1
2
0
27


HPV 70
0
0
0
0
0
0
0


HPV 71
−15
−10
11
−1
−10
1
−9


HPV 72
0
0
0
0
0
0
0


HPV 73
35
24
31
20
17
0
17


HPV 74
4
−3
−13
3
−3
5
−11


HPV 81
33
36
−12
7
−8
13
−10


HPV 82
0
0
0
0
0
0
0


HPV 83
−3
2
−14
−2
2
−9
4


HPV 84
4
−15
13
−22
−11
−16
11


HPV 85
0
0
0
0
0
0
0


HPV 86
6
391
10
26
19
24
4


HPV 87
14
−9
−12
5
12
10
−16


HPV 89
−1
6
3
−7
−11
−4
−4


HPV 90
5
19
−14
2
6
22
4


HPV 91
19
9
8
11
17
21
15


HPV 97
−12
10
9
3
28
1
2

















HPV
HPV
HPV
HPV
HPV
HPV
HPV
HPV
HPV


Sample
56
58
59
61
62
66
67
68
69





HPV 6
0
0
0
5
0
0
0
0
0


HPV 11
−3
14
2
−13
6
−4
5
−1   
−10


HPV 13
10
−6 
−15
−3
−11
−10
−19
16
−12


HPV 16
0
0
0
6
0
0
0
0
0


HPV 18
0
0
0
0
0
0
0
0
0


HPV 26
0
0
0
0
0
0
0
0
0


HPV 30
4
0
0
0
0
0
14
0
14


HPV 31
0
0
0
0
0
0
0
0
0


HPV 32
0
0
0
0
18
0
16
18
0


HPV 33
0
0
0
0
0
0
4
0
15


HPV 35
7
0
0
0
0
0
23
6
0


HPV 39
0
0
0
5
0
0
0
0
0


HPV 40
0
0
0
0
0
0
0
0
0


HPV 42
0
0
0
0
0
0
0
0
0


HPV 43
0
0
0
0
0
0
0
0
0


HPV 44
−13
−4
5
−8 
 −15
−12
−3
20
−9


HPV 45
0
0
0
0
0
0
0
0
0


HPV 51
0
0
0
0
0
0
0
0
0


HPV 52
−4
16
−5
−6
0
−5
−31 
−3
 5


HPV 53
0
0
0
0
0
0
0
0
0


HPV 54
0
0
0
0
0
0
0
0
0




















HPV
HPV
HPV
HPV
HPV
HPV
HPV
HPV
HPV


Sample
56
58
59
61
62
66
67
68
69





HPV 56
1638
0
0
0
0
0
0
0
0


HPV 58
0
1874
0
0
0
0
0
0
0


HPV 59
0
0
3405
0
0
0
0
0
0


HPV 61
0
0
0
3831
0
0
0
0
0


HPV 62
−11
−13
−6
2
2298
−2
−12
10
14


HPV 66
0
0
0
0
0
1258
0
0
0


HPV 67
0
0
0
0
0
0
1115
0
0


Hpv 68
6
−11
−18
15
−6
8
12
957
5


HPV 69
−10
7
−12
−1
−4
8
−8
21
1909


HPV 70
0
0
0
0
0
0
0
0
0


HPV 71
1 
−3                   
−1
−2
−17
20
−11
−19
−2


HPV 72
0
0
0
0
0
0
0
0
0


HPV 73
11
0
0
25
38
4
37
23
39


HPV 74
−10
−3
5
20
−4
3
15
8
1


HPV 81
−9
0
−27
3
−10
−10
47
47
4


HPV 82
0
0
0
0
0
0
0
0
0


HPV 83
15
−3
−9
9
−15
11
−6
8
2


HPV 84
−12
0
3
−2
1
−6
−16
17
−9


HPV 85
0
0
0
0
0
0
6
0
0


HPV 86
15
−2
−3
1
6
12
5
32
14


HPV 87
−7
−13
−8
−8
16
−13
7
31
3


HPV 89
−3
−3
−1
−5
4
15
7
7
−14


HPV 90
1
−6
2
11
6
5
−24
40
−10


HPV 91
11
3
−3
13
9
5
32
16
15


HPV 97
−3
−6
5
−16
−4
−9
−8
3
7



















HPV
HPV
HPV
HPV
HPV
HPV
HPV
HPV


Sample
70
71
72
73
74
81
82
83





HPV 6
1
2
0
0
0
0
2
0


HPV 11
−10
143
−10
2
−6
−9
−18 
−6


HPV 13
0
−11
254
4
26
5
−12  
−15


HPV 16
0
0
15
0
4
0
0
0


HPV 18
0
3
12
0
0
0
0
0


HPV 26
0
0
21
0
0
0
1
0


HPV 30
0
8
0
0
0
0
0
0


HPV 31
0
0
5
0
11
0
0
0


HPV 32
0
0
13
0
3
0
0
0


HPV 33
0
0
0
3
0
0
0
7


HPV 35
0
0
27
0
18
0
13
0


HPV 39
0
0
9
0
0
0
0
0


HPV 40
0
0
0
0
0
0
0
0


HPV 42
0
0
0
0
0
0
0
0


HPV 43
1
0
0
0
0
0
0
0


HPV 44
1
−18
−13
−7
−16
−15
−13
−13


HPV 45
0
0
0
0
0
0
0
0


HPV 51
0
0
0
0
0
0
0
0


HPV 52
−3
−16
147 
6
3
41
−7
−3


HPV 53
0
0
13
0
0
0
0
0


HPV 54
0
0
0
0
0
0
0
0






HPV
HPV
HPV
HPV
HPV
HPV
HPV
HPV


Sample
70
71
72
73
74
81
82
83





HPV 56
0
0
78
0
0
0
0
0


HPV 58
0
0
0
0
0
0
0
0


HPV 59
0
0
0
0
0
0
0
0


HPV 61
0
0
1
0
0
0
0
0


HPV 62
3
−6
−119
−10
−7
0
−10
−9


HPV 66
0
0
0
0
0
0
0
0


HPV 67
0
0
0
0
0
0
0
0


Hpv 68
6
−6
−106
9
10
−9
3
3


HPV 69
17
−8
−131
−3
16
20
−17
−5


HPV 70
3971
0
0
0
0
0
0
0


HPV 71
−5
1166
−229
−1
−17
−8
−3
−13


HPV 72
0
0
2213
0
0
0
0
0


HPV 73
23
18
20
1715
18
9
24
36


HPV 74
16
2
−127
−3
1044
−12
5
7


HPV 81
1
−16
115
−15
−14
2343
−10
−6


HPV 82
0
0
1
0
0
0
2259
0


HPV 83
−12
−11
−142
6
−6
−5
5
2625


HPV 84
12
−15
−82
−12
3
10
−2
11


HPV 85
0
0
0
0
0
0
0
0


HPV 86
6
28
−115
5
−2
8
8
2


HPV 87
−3
−8
−183
−1
7
14
4
2


HPV 89
6
−7
−68
−14
3
−5
0
−17


HPV 90
−16
3
−122
1
−4
6
−6
20


HPV 91
9
23
−51
16
9
7
17
−9


HPV 97
−4
−1
−73
2
−11
−7
−10
16



















HPV
HPV
HPV
HPV
HPV
HPV
HPV
HPV


Sample
84
85
86
87
89
90
91
97





HPV 6
0
0
0
11
0
0
0
17


HPV 11
4
−9
−9
−3 
−16
1
0
23


HPV 13
−8
−3
18
1
−12
2
1
28


HPV 16
6
4
0
0
0
0
0
28


HPV 18
9
0
0
0
0
0
0
−4


HPV 26
0
0
0
0
0
0
0
18


HPV 30
0
0
0
0
0
0
0
12


HPV 31
0
0
0
0
0
11
0
14


HPV 32
0
14
0
0
0
0
9
28


HPV 33
16
0
0
4
0
0
0
24


HPV 35
0
8
0
0
0
5
0
24


HPV 39
0
0
0
0
0
0
0
12


HPV 40
0
0
0
0
0
0
0
28


HPV 42
0
0
0
0
0
0
0
11


HPV 43
0
0
0
0
0
0
0
27


HPV 44
−4
8
11
−4
513
−5
8
28


HPV 45
0
0
0
0
0
0
0
0


HPV 51
0
0
0
0
0
0
0
16


HPV 52
−9
5
23
8
−6
7
−2
−8


HPV 53
0
0
0
0
0
0
0
21


HPV 54
0
0
0
0
0
0
0
17



















HPV
HPV
HPV
HPV
HPV
HPV
HPV
HPV


Sample
84
85
86
87
89
90
91
97





HPV 56
0
0
0
0
0
0
0
−25


HPV 58
0
0
0
0
0
0
0
1


HPV 59
0
0
0
0
0
0
0 
−22


HPV 61
0
0
0
0
0
0
0
−21


HPV 62
21
−15
−12
−4
2
−5
29
−20


HPV 66
0
0
0
0
0
0
0
−7


HPV 67
0
0
0
0
0
0
0
−5


Hpv 68
4
−11
−4
−5
−2
2
0
−11


HPV 69
8
4
−6
−1
11
−8
6
−28


HPV 70
0
0
0
0
11
0
0
1


HPV 71
10
−13
−4
−19
4
−18
−5
10


HPV 72
0
0
0
0
0
0
0
−13


HPV 73
14
15
24
9
4
36
27
−12


HPV 74
−7
−7
7
3
12
0
19
1


HPV 81
−20
−7
4
5
−3
7
−9
−18


HPV 82
0
0
0
0
0
0
0
−14


HPV 83
19
−17
6
6
−1
15
8
−7


HPV 84
2194
19
0
15
−13
−5
−15
−16


HPV 85
0
4605
0
0
0
0
0
−17


HPV 86
18
5
1720
35
12
13
2
−10


HPV 87
−3
−1
8
1828
12
7
1
0


HPV 89
9
12
−13
−10
3998
−27
1
−19


HPV 90
13
7
15
16
23
3613
5
−13


HPV 91
−8
6
14
21
24
31
2264
−16


HPV 97
6
2
−20
−3
−3
−7
−11
1267
















TABLE 5







Comparison of NML Luminex with direct sequencing for detection of


any HPV type


Direct sequencing












NML Luminex
Negative
Positive
Totals
















Positive
14
429
442



Negative
348
5
353



Totals
361
434
795

















TABLE 6







Distribution of HPV types as detected by


NML Luminex and direct sequencing










NML LUMINEX
Direct Sequencing
















% of


% of


HPV

% of
positive

% of
positive


type
n
types
samples
n
types
samples
















6
43
7.5%
5.4%
39
4.9%
4.9%


11
12
2.1%
1.5%
11
1.4%
1.4%


13
0
0.0%
0.0%
0
0.0%
0.0%


16
87
15.1%
10.9%
68
8.6%
8.6%


18
26
4.5%
3.3%
15
1.9%
1.9%


26
0
0.0%
0.0%
0
0.0%
0.0%


30
2
0.3%
0.3%
0
0.0%
0.0%


31
29
5.0%
3.6%
29
3.7%
3.6%


32
3
0.5%
0.4%
2
0.3%
0.3%


33
11
1.9%
1.4%
14
1.8%
1.8%


35
8
1.4%
1.0%
2
0.3%
0.3%


39
22
3.8%
2.8%
16
2.0%
2.0%


40
9
1.6%
1.1%
5
0.6%
0.6%


42
13
2.3%
1.6%
5
0.6%
0.6%


43
0
0.0%
0.0%
1
0.1%
0.1%


44
3
0.5%
0.4%
1
0.1%
0.1%


45
12
2.1%
1.5%
10
1.3%
1.3%


51
16
2.8%
2.0%
9
1.1%
1.1%


52
33
5.7%
4.2%
17
2.1%
2.1%


53
25
4.3%
3.1%
12
1.5%
1.5%


54
11
1.9%
1.4%
8
1.0%
1.0%


56
10
1.7%
1.3%
4
0.5%
0.5%


58
28
4.9%
3.5%
25
3.2%
3.1%


59
11
1.9%
1.4%
5
0.6%
0.6%


61
7
1.2%
0.9%
1
0.1%
0.1%


62
26
4.5%
3.3%
17
2.1%
2.1%


66
39
6.8%
4.9%
30
3.8%
3.8%


67
7
1.2%
0.9%
8
1.0%
1.0%


68
2
0.3%
0.3%
4
0.5%
0.5%


69
3
0.5%
0.4%
2
0.3%
0.3%


70
11
1.9%
1.4%
10
1.3%
1.3%


71
3
0.5%
0.4%
0
0.0%
0.0%


72
5
0.9%
0.6%
4
0.5%
0.5%


73
5
0.9%
0.6%
3
0.4%
0.4%


74
1
0.2%
0.1%
0
0.0%
0.0%


81
8
1.4%
1.0%
6
0.8%
0.8%


82
7
1.2%
0.9%
5
0.6%
0.6%


83
11
1.9%
1.4%
5
0.6%
0.6%


84
12
2.1%
1.5%
1
0.1%
0.1%


85
2
0.3%
0.3%
2
0.3%
0.3%


86
1
0.2%
0.1%
1
0.1%
0.1%


87
2
0.3%
0.3%
3
0.4%
0.4%


89
8
1.4%
1.0%
4
0.5%
0.5%


90
3
0.5%
0.4%
1
0.1%
0.1%


91
1
0.2%
0.1%
0
0.0%
0.0%


102
N/A
N/A
N/A
2
0.3%
0.3%
















TABLE 7







Comparison of NML Luminex vs Roche linear array for detection of


samples positive for any HPV type.


Roche Linear Array












NML Luminex
Negative
Positive
Totals
















Positive
46
394
440



Negative
424
16
440



Totals
470
410
880

















TABLE 8







Comparison between NML Luminex and Roche Linear array in the ability


to detect multiple HPV infections.










NML
Roche Linear



Luminex
Array















Positive for any type
435
405



Total HPV types detected
917
1111



Single infections
200
156



Multiple infections
235
249



2 types
122
87



3 types
49
59



4+ types
64
103

















TABLE 9







Comparison of HPV type distribution as detected by NML Luminex and


the Roche Linear Array method.














Roche




NML Luminex

Linear Array












n
%
n
%

















HPV06
48
5.2%
49
4.4%



HPV11
39
4.3%
45
4.1%



HPV13*
0
0.0%



HPV16
136
14.8%
137
12.3%



HPV18
47
5.1%
41
3.7%



HPV26
0
0.0%
3
0.3%



HPV30*
2
0.2%



HPV31
31
3.4%
48
4.3%



HPV32*
11
1.2%



HPV33
15
1.6%
16
1.4%



HPV35
30
3.3%
23
2.1%



HPV39
27
2.9%
39
3.5%



HPV40
10
1.1%
8
0.7%



HPV42
30
3.3%
38
3.4%



HPV43*
3
0.3%



HPV44
11
1.2%
16
1.4%



HPV45
34
3.7%
30
2.7%



HPV51
31
3.4%
57
5.1%



HPV52**
26
2.8%
74
6.7%



HPV53**
25
2.7%
53
4.8%



HPV54
14
1.5%
21
1.9%



HPV56
34
3.7%
29
2.6%



HPV58
27
2.9%
30
2.7%



HPV59
31
3.4%
44
4.0%



HPV61**
10
1.1%
27
2.4%



HPV62
14
1.5%
30
2.7%



HPV66
38
4.1%
38
3.4%



HPV67**
19
2.1%
6
0.5%



HPV68
13
1.4%
21
1.9%



HPV69
9
1.0%
9
0.8%



HPV70
30
3.3%
29
2.6%



HPV71
1
0.1%
1
0.1%



HPV72
7
0.8%
12
1.1%



HPV73**
6
0.7%
18
1.6%



HPV74*
12
1.3%



HPV81
12
1.3%
12
1.1%



HPV82
4
0.4%
11
1.0%



HPV83
12
1.3%
18
1.6%



HPV84**
18
2.0%
39
3.5%



HPV85*
5
0.5%



HPV86*
11
1.2%



HPV87
4
0.4%



HPV89**
18
2.0%
38
3.4%



HPV90*
12
1.3%



HPV91*
0
0.0%







*Type not detected by the Roche Linear Array



**Statistically significant difference (p < 0.05)













TABLE 10







Comparison of HPV type distribution as detected by NML Luminex and


the Roche Linear Array method when multiple infections with 4 or more


types are excluded.












NML

Roche




Luminex

Linear Array













Strain
n
%
n
%

















HPV06
26
4.9%
23
4.3%



HPV11
26
4.9%
30
5.6%



HPV13*
0
0



HPV16
86
16.1% 
90
16.9% 



HPV18
24
4.5%
17
3.2%



HPV26
0
  0%
0
  0%



HPV30*
1
0.2%



HPV31
24
4.5%
25
4.7%



HPV32*
5
0.9%
0
0.0%



HPV33
7
1.3%
5
0.9%



HPV35
14
2.6%
8
1.5%



HPV39
15
2.8%
16
3.0%



HPV40
8
1.5%
4
0.7%



HPV42
21
3.9%
21
3.9%



HPV43*
2
0.4%



HPV44
9
1.7%
5
0.9%



HPV45
12
2.2%
8
1.5%



HPV51
23
4.3%
29
5.4%



HPV52**
15
2.8%
34
6.4%



HPV53
19
3.6%
25
4.7%



HPV54
10
1.9%
12
2.2%



HPV56
20
3.7%
11
2.1%



HPV58
14
2.6%
11
2.1%



HPV59
11
2.1%
16
3.0%



HPV61
6
1.1%
14
2.6%



HPV62
10
1.9%
16
3.0%



HPV66
21
3.9%
19
3.6%



HPV67**
10
1.9%
2
0.4%



HPV68
9
1.7%
8
1.5%



HPV69
5
0.9%
5
0.9%



HPV70
15
2.8%
12
2.2%



HPV71
0
  0%



HPV72
4
0.7%
6
1.1%



HPV73
4
0.7%
9
1.7%



HPV74*
7
1.3%
0
0.0%



HPV81
4
0.7%
3
0.6%



HPV82
2
0.4%
4
0.7%



HPV83
8
1.5%
10
1.9%



HPV84
10
1.9%
17
3.2%



HPV85*
2
0.4%



HPV86*
5
0.9%



HPV87
1
0.2%



HPV89
11
2.1%
18
3.4%



HPV90*
9
1.7%



HPV91*
0
  0%

## Claims

1. A method of detecting and typing a human papillomavirus (HPV) type infection in a sample comprising:
a) providing a sample suspected of comprising at least one HPV type;
b) adding to the sample primers suitable for amplifying the L1 region of HPV;
c) incubating the sample under conditions suitable for DNA amplification;
d) adding a probe consisting of the nucleotide sequence of SEQ ID NO:46, said probe binding to only HPV type 97 under hybridization conditions, said probe further comprising a unique tag, said unique tag comprising a combination of two fluorescent dyes;
e) incubating said probe and said sample under conditions suitable for hybridization; and
f) detecting hybridization of said probe.

2. The method according to claim 1 wherein the unique tag is a combination of different ratios of red and infra-red fluorophores.

3. The method according to claim 1 wherein the primers comprise GP5+/GP6+.

4. The method according to claim 1 wherein the primers comprise GP5+/GP6+ and MY09/MY11.

5. The method according to claim 1 wherein at least one of the primers is exonuclease resistant.

6. The method according to claim 5 wherein exonuclease is added prior to step (d).