# Patent

## Title
Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids

## Abstract

Methods and materials for determining the presence of at least one nucleic acid in a sample are provided, said methods comprising (1) a purification step using sequence specific hybrid capture; (2) an amplification step; and (3) a detection step using two separate sequence-specific polynucleotide probes. Also provided are nucleic acids comprising SEQ ID NO: 1 to SEQ ID NO: 727 and nucleic acid probes and probe sets comprising the same.

## Background
REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 61/299,531, filed on Jan. 29, 2010, and U.S. Provisional Patent Application No. 61/326,067, filed on Apr. 20, 2010, each of which is incorporated herein by reference in its entirety.


FIELD OF INVENTION
The present disclosure relates to methods and compositions for purifying, detecting, and characterizing nucleic acids.
BACKGROUND
The identification of the presence or absence of specific nucleic acid sequences in a sample is a central part of many assays and tests used in the modern research lab and clinical setting. In the typical scheme, the nucleic acids from the sample are first separated from other macromolecules present in the sample by manipulating various physical properties. For example, nucleic acids typically bear a net negative charge at neutral pH, owing to the phosphodiester backbone. This property can be manipulated to separate nucleic acids from other macromolecules using anion exchange resins. As another example, differential solubility of nucleic acids compared to other macromolecules in certain solvents is used to extract nucleic acids from the sample. Numerous other such schemes exist. However, the amount of target nucleic acid relative to the total amount of nucleic acid purified typically is very low. Therefore, some type of amplification is necessary. Either the amount of specific nucleotide sequence(s) is increased by target amplification methods such as polymerase chain reaction (PCR) or the specific nucleotide sequence(s) is/are reacted with a detectable label and the signal from the label is amplified to detectable levels.
Unfortunately, these methods have limited utility. One limitation is that target-specific amplification methods such as PCR are inherently error-prone. For example, although the stringency of primer hybridization can be controlled, there nonetheless exists the potential for non-specific primer binding and primer-independent amplification, which can lead to false-positive results. Moreover, different sequences can amplify at different rates, resulting in amplification bias. As a result, quantitative analysis of multiple nucleic acid sequences in a single reaction often suffers from a lack of sensitivity. In addition, target nucleic acids that are present at low concentrations relative to other nucleic acids may be effectively “masked” from the polymerase, which could result in false-negative results. Other factors may exist that reduce both the specificity and sensitivity of such assays. Another limitation of PCR is that a relatively small fragment of the target is amplified. As a result, in case of mutations/deletions the assay may produce false-negative results.
Therefore, methods and compositions are needed for specific and sensitive isolation and analysis of at least one target nucleic acid segment containing at least one specific sequence.
SUMMARY
The present disclosure in aspects and embodiments addresses these various needs and problems by providing a method of detecting and genotyping at least one target nucleic acid and isolated nucleic acids useful for the same.
In one embodiment, an isolated nucleic acid is provided, having an overall length of not more than 100 nucleotides comprising, consisting essentially of, or consisting of at least one nucleotide sequence having at least 75-percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 727 and a complement thereof.
In an aspect, the isolated nucleic acid is capable of hybridizing under stringent conditions to a portion of a human papillomavirus (HPV) genome selected from the group consisting of: HPV2, HPV3, HPV6, HPV10, HPV11, HPV16, HPV18, HPV26, HPV27, HPV28, HPV29, HPV30, HPV31, HPV32, HPV33, HPV34, HPV35, HPV39, HPV42, HPV45, HPV51, HPV52, HPV53, HPV54, HPV56, HPV57, HPV58, HPV59, HPV64, HPV66, HPV67, HPV68, HPV69, HPV70, HPV73, HPV82, HPV84, HPV85, HPV86, HPV87, and HPV94.
In an aspect, the nucleic acid is capable of hybridizing under selective stringency conditions to an HPV gene selected from the group consisting of E6, E7, and L1.
In an aspect, the nucleic acid is not capable of hybridizing under stringent conditions to more than one human papillomavirus (HPV) genomes.
In an aspect, the nucleic acid is capable of hybridizing under stringent conditions to at least two of a group of human papillomavirus (HPV) genomes selected from the group consisting of: a) group A7, consisting of HPV18, HPV39, HPV45, HPV59, HPV68, HPV70, and HPV85; and b) group A9, consisting of HPV16, HPV31, HPV33, HPV35, HPV52, HPV58, and HPV67.
In another aspect, the nucleic acid is capable of hybridizing to a pair of HPV genomes selected from the group consisting of: a) HPV18 and HPV45; b) HPV39 and HPV68; c) HPV59 and HPV70; d) HPV70 and HPV85; e) HPV16 and HPV35; f) HPV31 and HPV35; g) HPV52 and HPV67; h) HPV33 and HPV58; i) HPV26 and HPV69; j) HPV51 and HPV82; k) HPV30 and HPV53; 1) HPV56 and HPV66; m) HPV34 and HPV73; and n) HPV6 and HPV11.
In an aspect, the isolated nucleic acid has at least 75-percent homology across its entire length to a portion of the human papillomavirus genome, the HPV selected from the group consisting of: HPV2, HPV3, HPV6, HPV10, HPV11, HPV16, HPV18, HPV26, HPV27, HPV28, HPV29, HPV30, HPV31, HPV32, HPV33, HPV34, HPV35, HPV39, HPV42, HPV45, HPV51, HPV52, HPV53, HPV54, HPV56, HPV57, HPV58, HPV59, HPV64, HPV66, HPV67, HPV68, HPV69, HPV70, HPV73, HPV82, HPV84, HPV85, HPV86, HPV87, and HPV94.
In an aspect, the isolated nucleic acid has at least 75-percent homology across its entire length to a portion of a gene selected from the group consisting of E6, E7, and L1.
In an aspect, the isolated nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 727, an RNA or DNA equivalent thereof, and a complement thereof.
In another aspect, a nucleic acid probe is provided, comprising an isolated nucleic acid as disclosed herein and optionally further comprising a detectable label and/or a ligand. In a further aspect, the nucleic acid probe is provided bound to a solid support.
In a further aspect, the nucleic acid probes as set forth above are provided as a part of a probe set.
In another aspect, a method of detecting a target nucleic acid in a sample comprising non-target nucleic acids is provided, said method comprising:

    (a) purifying the target nucleic acid from the sample by a method comprising:
    
        (i) contacting the sample with at least one purification probe, wherein at least a portion of the nucleic acid probe hybridizes to the at least one target nucleic acid to form a DNA:RNA hybrid;
        (ii) immobilizing the DNA:RNA hybrid to a first solid support by a method comprising contacting the DNA:RNA hybrid with at least a first antibody capable of binding to the DNA:RNA hybrid, wherein the antibody is bound to or adapted to be bound to the first solid support; and
        (iii) separating the first solid support from the sample to generate at least one purified target nucleic acid;
    
    
    b. genotyping the purified target nucleic acid by a method comprising:
    
        (i) amplifying at least a portion of the purified target nucleic acid to generate an amplicon, such as by an isothermal amplification, such as whole genome amplification;
        (ii) immobilizing the amplicon to a second solid support by a method comprising contacting the amplicon with at least one immobilization probe, wherein:
        
            (α) the immobilization probe is bound to or adapted to be bound to the second solid support; and
            (β) at least a portion of the immobilization probe hybridizes the at least one target nucleic acid;
        
        
        (iii) contacting the immobilized amplicon with at least one detection probe, wherein the at least a portion of the detection probe hybridizes to the at least one target nucleic acid to generate a detection complex; and
        (iv) detecting at least a first detectable signal generated by the detection complex, wherein the detectable signal indicates the genotype of the target nucleic acid.
    
    


In another aspect, the purified nucleic acid is fragmented before amplification.
In another aspect, the second solid support generates the first detectable signal.
In another aspect, a plurality of distinct purified target nucleic acids are generated.
In another aspect, the plurality of purified target nucleic acids is contacted with a plurality of immobilization probes, wherein each of the plurality of immobilization probes is specific for a distinct purified target nucleic acid.
In another aspect, at least two of the plurality of immobilization probes are specific for the same purified target nucleic acid.
In another aspect, at least two of the plurality of immobilization probes are specific for different regions of the same purified target nucleic acid.
In another aspect, a plurality of distinct second solid supports are used, wherein: (α) each second solid support comprises at least one immobilization probe specific for a single target nucleic acid and does not comprise any immobilization probes specific for any other of the plurality of target nucleic acids; and (β) each solid support generates a unique first detectable signal that indicates the genotype of the target nucleic acid.
In another aspect, a second detectable signal is generated that indicates immobilization of the amplicon to the second solid support.
In another aspect, the detection probe comprises a detectable label that generates the second detectable signal.
In another aspect, the second detectable signal further indicates the genotype of the target nucleic acid.
In another aspect, the second detectable signal further indicates the quantity of amplicon immobilized to each solid support.
In another aspect, the first detectable signal indicates a genotype of a human papillomavirus (HPV) selected from the group consisting of: high-risk HPV (HR-HPV) types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82; and low-risk HPV (LR-HPV) types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91.
In another aspect, at least one of the purification probe, immobilization probe, and/or detection probe comprises an isolated nucleic acid having an overall length of not more than 100 nucleotides and comprising a sequence having at least 75-percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 727, a DNA or RNA equivalent thereof, and a complement thereof.
In another aspect, the purification probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 727 and a complement thereof.
In another aspect, the immobilization probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 344 to SEQ ID NO: 727 and a complement thereof.
In another aspect, the detection probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 727 and a complement thereof.
In another aspect, a method is provided comprising:

    a. a purifying step comprising:
    
        generating a double-stranded nucleic acid hybrid of the at least one target nucleic acid by hybridizing the at least one target nucleic acid to a hybrid probe set comprising at least a first nucleic acid probe specific for the at least one target nucleic acid;
        immobilizing the double-stranded nucleic acid hybrid to a first solid support through by contacting the double-stranded nucleic acid hybrid with at least a first antibody capable of binding to the double-stranded nucleic acid hybrid and binding the at least a first antibody to the first solid support; and
        separating the double-stranded nucleic acid hybrid from the sample to generate at least one purified nucleic acid;
    
    
    b. an amplifying step, wherein at least a portion of the at least one purified nucleic acid is amplified to generate amplified nucleic acids; and
    c. a genotyping step comprising:
    
        immobilizing the amplified nucleic acids to at least a second solid support by hybridizing the amplified nucleic acids to an immobilization probe set comprising at least one polynucleotide probe specific for the at least one target nucleic acid; and
        detecting the presence of the at least one target nucleic acid with a detection probe set comprising at least one polynucleotide probe specific for the at least one target nucleic acid.
    
    


In a further aspect, the amplification step comprises an isothermal amplification.
In a further aspect, the amplification step comprises whole genome amplification.
In a further aspect, the amplified nucleic acids are fragmented before the genotyping step.
In a further aspect, the immobilization probe set is bound to a plurality of solid supports placed in suspension.
In a further aspect, the plurality of solid supports is detectably labeled.
In a further aspect, the methods described herein are adapted to detect the presence of a plurality of target nucleic acids.
In a further aspect, the immobilization probe set comprises at least one probe specific for each of the plurality of target nucleic acids.
In a further aspect, the immobilization probe set consists essentially of two probes specific for each of the plurality of target nucleic acids.
In a further aspect, the two probes specific for of the plurality of target nucleic acids bind to distinct regions of the variants.
In a further aspect, each solid support of the plurality of solid supports contains only probes specific for one nucleic acid of the plurality of target nucleic acids, such that only the one nucleic acid of the plurality of target nucleic acids will bind to each of the plurality of solid supports.
In a further aspect, each of the plurality of solid supports is detectably labeled such that a solid support specific for a first nucleic acid of the plurality of target nucleic acids bears a different detectable label than a solid support specific for a second nucleic acid of the plurality of target nucleic acids.
In a further aspect, the detection probe set is detectably labeled.
In a further aspect, the detectable label of each of the plurality of solid supports is used to indicate the identity of the target nucleic acid bound thereto; and the detectable label of the detection probe set is used to indicate the relative amount of the target nucleic acid bound to each solid support.
In a further aspect, the at least one target nucleic acid is an human papillomavirus (HPV) nucleic acid.
In a further aspect, the HPV nucleic acid is selected from the group consisting of: high-risk HPV (HR-HPV) types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82; and low-risk HPV (LR-HPV) types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91.
In a further aspect, a plurality of HPV nucleic acids are detected.
In a further aspect, the plurality of HPV nucleic acids comprises, consists, or consists essentially of: HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82, or any subset thereof; and/or LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91, or any subset thereof.
In a further aspect, the methods disclosed herein are adapted such that 59 high- and LR-HPV types can be detected and identified in a single reaction.
In another aspect, a kit for genotyping a nucleic acid is provided comprising: (a) an isolated nucleic acid as disclosed herein; (b) a nucleic acid polymerase; (c) a primer; (d) a first solid support; (e) an anti-DNA:RNA hybrid antibody bound to or adapted to be bound to the first solid support; and (f) a detectably labeled second solid support.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a graphical representation of conventional amplification results and how human genomic DNA reduces the amplification of the desired target; and FIG. 1B is a graphical representation of hybrid capture and amplification results and how the desired target's amplification is not reduced by human genomic DNA.
FIG. 2 is a graph showing the results of a 20-plex reaction detecting quadruple HPV infections.
FIG. 3A is a graphical representation of target detection results at various amplicon volumes; and 3B is a graphical representation of target detection results after overnight amplification.
FIGS. 4A and 4B are graphical representations of target detection results for two HPV types.
FIG. 5 is a graphical representation of multiplex experiment results testing for 26 HPV types and all HR-HPV types.
FIG. 6 is a data table displaying S/N values of multiplex experiment results testing for 26 HPV types and all HR-HPV types.
FIG. 7 is a graphical representation of detection results of a single HPV type infection.
FIG. 8 is a graphical representation of detection results of a single HPV type infection.
FIG. 9 is a graphical representation of detection results of a quadruple HPV type infection.
FIG. 10 is a graphical representation of detection results of a double HPV type infection.
FIG. 11 is a schematic illustrating hybrid capture, whole genome amplification, and detection of the target nucleic acids.



DETAILED DESCRIPTION
The present disclosure covers methods, compositions, reagents, and kits for determining the presence of at least one target nucleic acid in a sample. The methods, compositions, reagents, systems, and kits may be used for clinical diagnostic purposes, including but not limited to the detection and identification of pathogenic organisms and the detection of a genetic predisposition to a particular disease.
I. Samples and Sample Preparation
A. Samples
Any sample may be used as a starting point, including, without limitation, a specimen or culture (e.g., cellular, microbiological and viral cultures) including clinical and laboratory biological and environmental samples. Biological samples may be from an animal, including a human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items.
Exemplary biological samples include, but are not limited to, cervical epithelial cells (e.g., a sample obtained from a cervical swab), adenoid cells, anal epithelial cells, blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum and semen.
In an aspect, the biological sample is collected and stored in a collection medium. The collection medium has several functions including as a preservative medium to preserve nucleic acids and inhibit nucleases to prevent degradation of nucleic acids prior to analysis. In one aspect, the collection medium is detergent-based. Without being limited, exemplary collection media include those found in U.S. Patent Publication No. US 2010-0105060 A1 and U.S. Patent Publication No. US 2010-0159463 A1, both of which are hereby incorporated by reference in their entirety.
In one aspect the detergent-based collection medium comprises, consists essentially of, or consists of 1.0% NP-40, 0.25% sodium deoxycholate, 50 mM Tris-HCl, 25 mM EDTA, 150 mM NaCl and 0.05% sodium azide. In another aspect the detergent-based collection medium comprises, consists essentially of, or consists of about 0.5% to about 2.0% NP-40, about 0.10% to about 0.40% sodium deoxycholate, about 25 mM to about 75 mM Tris-HCl, about 10 mM to about 50 mM EDTA, about 50 mM to about 200 mM NaCl, and about 0.01% to about 0.10% sodium azide. In other aspects the detergent-based collection medium comprises, consists essentially of, or consists of about 0.8% to about 1.5% NP-40, about 0.20% to about 0.40% sodium deoxycholate, about 30 mM to about 60 mM Tris-HCl, about 20 mM to about 40 mM EDTA, about 100 mM to about 200 mM NaCl, and about 0.025% to about 0.075% sodium azide. In yet another aspect the detergent-based collection medium comprises, consists essentially of, or consists of about 0.9% to about 1.2% NP-40, about 0.20% to about 0.30% sodium deoxycholate, about 30 mM to about 60 mM Tris-HCl, about 20 mM to about 30 mM EDTA, about 100 mM to about 150 mM NaCl, and about 0.04% to about 0.06% sodium azide.
In an aspect, the collection medium comprises, consists essentially of, or consists of NP-40 and EDTA. In another aspect, the collection medium comprises, consists essentially of, or consists of NP-40, EDTA, and sodium azide. In one aspect, the collection medium comprises, consists essentially of, or consists of sodium deoxycholate, EDTA, and sodium azide. In an aspect, the collection medium comprises, consists essentially of, or consists of about NP-40, sodium deoxycholate, EDTA, and sodium azide. In an aspect, the collection medium comprises, consists essentially of, or consists of NP-40, sodium deoxycholate, Tris-HCl, EDTA, and sodium azide.
In another aspect, the collection medium comprises, consists essentially of, or consists of 0.5% to about 2.0% NP-40 and 10 mM to about 50 mM EDTA. In another aspect, the collection medium comprises, consists essentially of, or consists of 0.5% to about 2.0% NP-40, 10 mM to about 50 mM EDTA, and about 0.01% to about 0.10% sodium azide. In one aspect, the collection medium comprises, consists essentially of, or consists of about 0.10% to about 0.40% sodium deoxycholate, 10 mM to about 50 mM EDTA, and about 0.01% to about 0.10% sodium azide. In an aspect, the collection medium comprises, consists essentially of, or consists of about 0.5% to about 2.0% NP-40, about 0.10% to about 0.40% sodium deoxycholate, 10 mM to about 50 mM EDTA, and about 0.01% to about 0.10% sodium azide. In an aspect, the collection medium comprises, consists essentially of, or consists of about 0.5% to about 2.0% NP-40, about 0.10% to about 0.40% sodium deoxycholate, about 25 mM to about 75 mM Tris-HCl, about 10 mM to about 50 mM EDTA, and about 0.01% to about 0.10% sodium azide. In certain aspects, the medium comprises or consists essentially of 1% NP-40, 0.25% sodium deoxycholate, 50 mM Tris-HCl, 25 mM EDTA, 150 mM NaCl and 0.09% sodium azide. This medium is often referred to herein as Digene Collection Medium or DCM.
Samples may be collected in other known collection mediums and can be used in the methods described herein. Examples of other collection media include PRESERVCYT, SUREPATH, urine, and STM (Sample/Specimen Transport Medium). Samples collected in some of these media may require processing before the nucleic acids in the samples can be detected and analyzed. Various methods of processing samples (also known as preparing the samples) are known in the art. For example, cervical cell samples collected for cytological analysis in medium such as PRESERVCYT may be combined with a detergent-based lysis buffer followed by the addition of magnetic beads comprising nucleic acid binding surfaces.
In another aspect, the sample may comprise, consist, or consist essentially of nucleic acids that have been extracted from a biological sample. Numerous methods are known for extracting nucleic acids from a biological or environmental sample, including but not limited to: phenol/chloroform extraction; anion exchange chromatography; cesium chloride gradient ultracentrifugation; size exclusion chromatography; and silca/chaotropic salt extraction. Extracted nucleic acids may be further separated according to size by gel electrophoresis and extracted from the gel if samples comprising specific nucleic acid sizes are desired.
B. Target Nucleic Acids
As noted above, the methods disclosed herein relate to the detection and genotyping of at least one target nucleic acid in a sample. The at least one target nucleic acid may be DNA or RNA or both DNA and RNA and can be single-stranded, double-stranded, or partially single-stranded. The at least one target nucleic acid can be contained within a larger nucleic acid. Detection of either the at least one target nucleic acid or the larger nucleic acid comprising the at least one target nucleic acid is contemplated by this disclosure.
The at least one target nucleic acids may include, without limitation, nucleic acids found in specimens or cultures (e.g., cellular, microbiological and viral cultures) including biological and environmental samples. The at least one target nucleic acids may be found in biological samples from an animal, including a human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. At least one target nucleic acids may be found in environmental samples and include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items.
The at least one target nucleic acids found in biological samples include, but are not limited to, cervical samples (e.g., a sample obtained from a cervical swab) or cervical cell samples, adenoid cells, anal epithelial cells, blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, urine and semen. The at least one target nucleic acids may be from other viral, bacteria, mycobacteria or plasmodia, such as cytomegalovirus (CMV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), H1N1, Neisseria gonorrhoeae (GC), Chlamydia trachomatis (CT), Trichomonas vaginalis, Staphylococcus aureus, mycobacterium tuberculosis, SARS-associated coronavirus or influenza.
In an aspect the at least one target nucleic acids are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, at least 99%, or 100% identical to nucleic acids associated with any one of cervical samples (e.g., a sample obtained from a cervical swab) or cervical cell samples, adenoid cells, anal epithelial cells, blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, urine and semen, other viral, bacteria, mycobacteria or plasmodia, for example cytomegalovirus (CMV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), H1N1, Neisseria gonorrhoeae (GC), Chlamydia trachomatis (CT), Trichomonas vaginalis, Staphylococcus aureus, mycobacterium tuberculosis, SARS-associated coronavirus or influenza.
In one aspect, the at least one target nucleic acid is an HPV nucleic acid. In another aspect, the HPV nucleic acid is HPV DNA of a HR-HPV type. In another aspect, the HPV nucleic acid is HPV RNA of a LR-HPV type. In another aspect the at least one target nucleic acids are any one of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 or any one of LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91.
In another aspect, a plurality of target nucleic acid is targeted. In one aspect, the plurality of target nucleic acids consists of a set of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleic acids having distinct nucleotide sequences. Any set of nucleic acids to be targeted can be used. In one aspect, the plurality of target nucleic acids is selected such that each is related to the others. By way of example and not limitation, the set of nucleic acids can be: structurally related to one another (for example, members of a gene family); functionally related to one another (for example, nucleic acids encoding proinflammatory cytokines); phylogenetically related to one another (for example, nucleic acids specific for different members of a family of viruses, such as HPV-family viruses); related by virtue of differential expression in a different cell or tissue type (for example, macrophage-associated nucleic acids and prostate-associated nucleic acids) or disease states (cervical cancer associated nucleic acids). In another aspect, the set of nucleic acids is unrelated.
In one aspect, a set of target nucleic acids comprises, consists, or consists essentially of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82, or any subset thereof. In another aspect, a set of target nucleic acids comprises, consists, or consists essentially of LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91, or any subset thereof. In another aspect a set of target nucleic acids comprises, consists, or consists essentially of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82, or any subset thereof; and LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91, or any subset thereof. In another aspect, the at least one target nucleic acid is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, at least 99%, or 100% identical to nucleic acids associated with any one of HPV, genetic variants of HPV, HPV DNA of a HR-HPV type, or HPV RNA of a HR-HPV type. In another aspect the at least one target nucleic acids are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, at least 99%, or 100% identical to nucleic acids associated with any one of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 or any one of LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91.
As noted previously, the at least one target nucleic acid may be DNA or RNA. When the at least one target nucleic acid is DNA, the probe can be RNA and when the at least one target nucleic acid is RNA, the probe is can be DNA. However, a DNA probe can be used with DNA at least one target nucleic acid and an RNA probe can be used with RNA at least one target nucleic acid.
C. Sample Preparation
After the sample is collected in a collection medium as described above, the sample may be treated with a denaturation reagent to render the at least one target nucleic acid accessible to hybridization. In one aspect, the sample is denatured with an alkaline solution. Without being limited, suitable alkali include NaOH and KOH.
Alkaline treatment of protein effectively homogenizes the specimen to ensure reproducibility of analysis results for a given sample. It can also reduce the viscosity of the sample to increase kinetics, homogenize the sample, and reduce background by destroying any endogenous single stranded RNA nucleic acids, DNA-RNA hybrids or RNA-RNA hybrids in the sample. It also helps inactivate enzymes such as RNases and DNases that may be present in the sample. One skilled in that art would appreciate that if RNA is the at least one target nucleic acid (as opposed to DNA), different reagents may be preferable including, but not limited to phenol extraction and TCA/acetone precipitation, and guanidinium thiocyanate-phenol-chloroform extraction.
Other methods of denaturation may be employed such as utilizing a heating step, for example, heating the sample to about 95° C. to separate the strands of nucleic acid. Enzymes such as helicase may be used as well.
II. Purification
In the typical assay to detect nucleic acids in a sample, a large, non-specific extraction of nucleic acids is performed. The user then attempts to amplify or detect the target nucleic acid in the presence of this large pool of non-specific nucleic acids. However, the non-specific pool of nucleic acids oftentimes interferes with the amplification or detection step desired, particularly when the target nucleic acid is at a low concentration compared to the non-specific nucleic acids. The presently disclosed methods therefore separate the target nucleic acid from the non-specific pool of nucleic acid before the detection is performed by: (1) hybridizing a sequence specific polynucleotide purification probe to the target nucleic acid to form a double-stranded nucleic acid hybrid; (2) complexing the double-stranded nucleic acid hybrid to at least one molecule that specifically binds to double-stranded nucleic acid hybrids; and (3) capturing the complex to a solid support.
A. Hybridization of Probes
After the sample comprising the nucleic acid is prepared for hybridization, it is contacted with at least one polynucleotide hybrid probe under a condition sufficient for the one or more polynucleotide hybrid probes to hybridize to the at least one target nucleic acid in the sample to form a double-stranded nucleic acid hybrid. The at least one polynucleotide hybrid probe can be full length, truncated, or synthetic DNA or full length, truncated, or synthetic RNA. If the at least one target nucleic acid is DNA, then the at least one polynucleotide hybrid probe may be RNA and if the at least one target nucleic acid is RNA, then the probe may be DNA.
In one aspect, a single polynucleotide probe is used to purify the target nucleic acid. The single polynucleotide probe may be specific for only a single target nucleic acid or may be designed so as to hybridize to a plurality of target nucleic acids under stringent conditions. By way of example and not limitation, a polynucleotide probe may be designed against a highly conserved region of nucleic acids encoding a specific gene product, such that the polynucleotide probe would be expected to hybridize under stringent conditions to substantially all nucleic acids encoding that gene product.
In another aspect, a plurality of polynucleotide probes is used to purify the target nucleic acid. The plurality of polynucleotide probes may be specific for only a single target nucleic acid or may be specific for a plurality of target nucleic acids. By way of example and not limitation, a plurality of polynucleotide probes specific for a single target nucleic acid may be generated by fragmenting the target nucleic acid. In one aspect, at least one polynucleotide hybrid probes is provided for each target nucleic acid. In another aspect, at least two polynucleotide hybrid probes are provided for each target nucleic acid.
In an aspect, the polynucleotide hybrid probe is capable of hybridizing or binding to nucleic acids at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, at least 99%, or 100% identical to nucleic acids associated with HPV, genetic variants of HPV, HPV DNA of a HR-HPV type, or HPV RNA of a HR-HPV type, or any one of one of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 or any one of LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91. In another aspect, the probe is complementary to HPV, genetic variants of HPV, HPV DNA of a HR-HPV type, HPV RNA of a HR-HPV type, or any one of one of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 or any one of LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91.
In another aspect, a plurality of polynucleotide hybrid probes is provided, the plurality being selected to hybridize to and purify each of a set of target nucleic acids. In one aspect, the plurality of polynucleotide hybrid probes is capable of hybridizing to each nucleic acid of a set of target nucleic acids consisting of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 nucleic acids, or any subset thereof. In one aspect, the plurality of polynucleotide hybrid probes is capable of hybridizing to each nucleic acid of a set of target nucleic acids consisting of LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91, or any subset thereof. In one aspect, the plurality of polynucleotide hybrid probes is capable of hybridizing to each nucleic acid of a set of target nucleic acids consisting of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82, or any subset thereof; and LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91, or any subset thereof.
If the at least one target nucleic acid was denatured using an alkaline treatment, the one or more polynucleotide probes may be diluted in a probe diluent that also can act as a neutralizing hybridization buffer (to neutralize the basic denaturation reagent).
The probe diluent used for DNA or RNA probes will differ due to the different requirements necessary for DNA versus RNA stability. For example, if the probes are RNA, it is preferable to neutralize the sample first and then add the probe or alternatively, add the RNA probe and neutralizing agent (probe diluent) to the sample at the same time as excessive alkalinity can destroy RNA. The probe diluent can be used to dissolve and dilute the probe and also help restore the sample to about a neutral pH, e.g., about pH 6 to about pH 9, to provide a more favorable environment for hybridization. Sufficient volume of probe diluent, preferably one-half volume of the sample, may be used to neutralize the base-treated sample.
For full length probes, a heated alkaline solution may be added to the sample, then probe diluent may be added to the sample at room temperature, and then the sample may be reheated. Such a process can inhibit secondary structure from forming. Antibodies tend to irreversibly bind to structures with secondary structure. When using non-full length probes such as truncated or synthetic probes, heating the solutions or sample may not be necessary because secondary structures issues are not present. In an aspect, the sample is not heated when used with truncated or synthetic probes.
After treatment with the denaturation reagent, an aliquot of neutralization buffer, in an aspect the probe diluent described, in which the one or more probes are dissolved, can be added to the sample under appropriate conditions to allow hybridization or binding of the probe and the at least one target nucleic acid to occur. The neutralization buffer may contain a single buffering salt. In an aspect, the neutralization buffer does not contain more than a single buffering salt. The hybridization condition is sufficient to allow the one or more polynucleotide probes to anneal to a corresponding complementary nucleic acid sequence, if present, in the sample to form a double-stranded nucleic acid hybrid.
Hybridization conditions suitable for the particular probes and diluents described herein are employed. For example, the probes and sample nucleic acids can be incubated for a hybridization time, preferably at least about 5 to about 30 minutes, about 5 to about 20 minutes, or from about 7 to about 15 minutes, or about 10 minutes, as well as any number within the recited ranges sufficient to allow the one or more polynucleotide probes to anneal to a corresponding complementary nucleic acid sequence. The hybridization condition can include a hybridization temperature of at least about 65° C., about 68.5° C., and about 67° C. to about 70° C., as well as any number within the recited ranges. For a given at least one target nucleic acid and a given probe, one of ordinary skill in the art can readily determine desired hybridization conditions by routine experimentation. One of ordinary skill in the art will further appreciate that the time and temperature of hybridization must be optimized, one with respect to the other. Thus, higher hybridization temperatures may be carried out for shorter periods of time and vice versa. Without being limited, stringent hybridization conditions may be controlled by increasing the temperature, increasing the ionic conditions to above 0.5M (for example, NaCl), or reducing the concentration of PAA. As a non-limiting example, stringent hybridization conditions may include performing a hybridization reaction at elevated temperatures, such as of at least about 65° C., at least about 68.5° C., between about 67° C. to about 70° C., and between about 69° C. to about 70° C. Stringent hybridization conditions may also include elevated temperatures, such as of at least about 65° C., at least about 68.5° C., and between about 67° C. to about 70° C. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995), incorporated by reference in its entirety.
For present purposes, “stringent conditions” encompass conditions under which hybridization will only occur if there is 25% mismatch or less between the hybridization molecule and the target sequence. “Stringent conditions” may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, “moderate stringency” conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of “medium stringency” are those under which molecules with more than 15% mismatch will not hybridize, and conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize. Conditions of “very high stringency” are those under which sequences with more than 6% mismatch will not hybridize. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are also discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11, herein incorporated by reference in its entirety.
In an aspect, the hybridization/capture step is completed at 50° C. in about 15-25 minutes; at 50° C. in about 20-25 minutes; or at 50° C. in about 22.5 minutes.
In one aspect, the sample is suspended in collection medium, the at least one target nucleic acid is denatured with a denaturation reagent, and hybridized to nucleic acid probes suspended in a neutralizing buffer. In another aspect the neutralizing buffer is the probe diluent of the present invention. In another aspect, the probe diluent comprises 2.2 M BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), 2.6% polyacrylic acid, 0.7 N NaOH and 0.05% sodium azide.
B. Complexing and Capturing the Double-Stranded Nucleic Acid Hybrid
After the probes are allowed to hybridize to the at least one target nucleic acid and form a double-stranded nucleic acid hybrid, the hybrid is captured by a molecule that is specific for the double-stranded nucleic acid hybrid. Molecules specific for the double-stranded nucleic acid hybrids include, but are not limited to, monoclonal antibodies, polyclonal antibodies, proteins such as but not limited to RNAse H, nucleic acids including but not limited to aptamers, or sequence specific nucleic acids. Aptamers are short stretches of random sequences that are successively selected from a library of sequences by hybridizing to a target, amplifying the hybridized aptamers, and repeating the selection process.
In one aspect the molecule specific for the double-stranded nucleic acid hybrid is captured by an antibody, known as an anti-hybrid antibody. In another aspect, the anti-hybrid antibodies are immobilized onto a support before the double-stranded nucleic acid hybrid is captured. Methods of immobilizing antibodies to solid supports are well known in the art. By way of example and not limitation, the antibodies can be covalently linked to the solid support. As another example, the antibody can be adsorbed onto the adsorption, for example, protein-protein interactions, protein-G beads, biotin-streptavidin interaction, EDAC to link to a carboxyl or tosyl group, etc., or hybridization directly onto the solid support using, for example, sequence specific nucleic acids in an affinity column.
In another aspect, the anti-hybrid antibodies may be complexed with the double-stranded nucleic acid hybrid before being immobilized on the solid support. By way of example and not limitation the anti-hybrid antibody may be conjugated with a biotin label, while the support may be conjugated with a streptavidin moiety. Anti-hybrid antibody/double-stranded nucleic acid-hybrid complexes can then be allowed in the absence of the solid support. When the solid support is added to the reaction mixture, the anti-hybrid antibody/double-stranded nucleic acid-hybrid complexes will be immobilized to the solid support by virtue of the interaction between the biotin conjugate and the streptavidin moiety.
Supports include but are not limited to beads; magnetic beads, including paramagnetic, diamagnetic, ferromagnetic, ferromagnetic, and diamagnetic beads, columns, plates, filter paper, polydimethylsiloxane (PDMS); dipsticks; coated tubes, plates, and dishes; and resin columns. Any support can be used as long as it allows extraction of the liquid phase and provides the ability to separate out bound and unbound antibodies. Paramagnetic beads are particularly useful in that they can be left in the solution and the liquid phase can be extracted or decanted, if a magnetic field is applied to immobilize the beads. Beads that are small and have a high surface area are preferable, such as beads about 1 μm in diameter. Other beads that employ charge switching or silica capture (as opposed to magnetic fields) may be used as well.
The hybrids are incubated with the anti-hybrid antibody attached to the support for a sufficient amount of time to allow capture of the double-stranded nucleic acid hybrids by the immobilized anti-hybrid antibodies. In an aspect, the support is a bead.
The anti-hybrid antibody may be monoclonal or polyclonal. In one aspect the antibody is monoclonal. In one aspect, the antibody is coupled to the support by a 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) linker. In one aspect, the support is a polystyrene bead. In an aspect, the support or bead coupled to the antibody is diluted in a bead dilution buffer. The bead dilution buffer is helpful in minimizing protein denaturation on the bead. One example of a bead dilution buffer comprises 6% casein, 100 mM Tris-HCl, 300 mM NaCl, and 0.05% sodium azide.
In an aspect, the beads coated with the anti-hybrid antibody are incubated with the sample at about 67° C. to about 70° C. for about 30 minutes. In another aspect, the beads and sample are incubated at about 68° C. to about 69° C. for about 30 minutes. In yet another aspect, the beads and sample are incubated at about 68.5° C. for 30 minutes. The incubation time can range from about 5 minutes to about 60 minutes, from about 15 minutes to about 45 minutes, from about 20 minutes to about 40 minutes, or any number within the recited ranges, and is generally inversely proportional to the temperature. It will be understood by those skilled in the art that the incubation time, temperature and/or shaking conditions can be varied to achieve alternative capture kinetics as desired.
Following capture of the at least one target nucleic acid/probe hybrid as described above, the captured hybrid may be separated from the rest of the sample by washing away of non-captured nucleic acids.
III. Amplification
Once the at least one target nucleic acid is purified, it is amplified. Amplification is performed at this time to increase the sensitivity of the method by increasing the amount of the at least one target nucleic acid.
Nucleic acid amplifications can be broadly separated into two categories: temperature cycled amplifications and isothermic amplifications.
In temperature cycled amplifications, the temperature typically is raised above the melting point of the target nucleic acid to “melt” any double stranded portions, and then lowered to a point at which oligonucleotide primers anneal with single stranded portion of the target nucleic acid, then raised again to a temperature at which the primers remain annealed and the polymerase is active.
In isothermic amplifications, an agent is added to the reaction mixture to permit amplification without temperature cycling. For example, in helicase-dependant amplification (“HDA”), an enzyme having helicase activity is added to the amplification mixture. As used herein, “helicase” or “an enzyme with, or having, helicase activity” refers to any enzyme capable of unwinding a double stranded nucleic acid. The helicase functions to unwind double stranded nucleic acids, thus obviating the need for repeated melting cycles. Examplary helicases include E. coli helicase I, II, III, & IV, Rep, DnaB, PriA, PcrA, T4 Gp41 helicase, T4 Dda helicase, T7 Gp4 helicases, SV40 Large T antigen, yeast RAD. Additional helicases that may be useful include RecQ helicase, thermostable UvrD helicases from T. tengcongensis and T. thermophilus, thermostable DnaB helicase from T. aquaticus, and MCM helicase from archaeal and eukaryotic organisms. As another example, in nick-initiated amplification (“NIA”), a nick-inducing agent is used to induce breaks in the phosphodiester bonds of the nucleic acid backbone. A polymerase having strand displacement activity can then initiate amplification at the site of the nick, using one strand of the nucleic acid as a primer and the other strand as a template. As used herein, “nick-inducing agent” refers to any enzymatic or chemical reagent or physical treatment that introduces breaks in the phosphodiester bond between two adjacent nucleotides in one strand of a double-stranded nucleic acid. Examples of nick-inducing enzymes include Bpu10 I, BstNB I, Alw I, BbvC I, BbvC I, Bsm I, BsrD, and E. coli endonuclease I.
The amplification in the disclosed methods can be either a temperature cycled amplification or an isothermic amplification. Exemplary methods of amplification include, but are not limited to: polymerase chain reaction (“PCR”), reverse transcriptase (“RT”) reaction, RT-PCR, HDA, RT-HDA, thermophilic helicase-dependent amplification (“tHDA”), RT-tHDA, whole genome amplification (“WGA”), RT-WGA, ligase chain reaction (“LCR”), RT-LCR, NIA, and RT-NIA.
Amplification reactions can further be separated into sequence-dependent or sequence-independent amplifications.
“Sequence-dependent amplification” refers to amplification of a target sequence relative to non-target sequences present in a sample with the use of target-specific primers. As used herein, “target-specific primer” refers to a single stranded nucleic acid capable of binding to a pre-determined single stranded region on a target nucleic acid to facilitate polymerase dependent replication of the target nucleic acid to be selectively amplified.
In one aspect, the amplification is a sequence-specific amplification. In another aspect, a pair of target-specific primers, one hybridizing to the 5′-flank of a target sequence within each target nucleic acid and the other hybridizing to the 3′-flank of the target sequence, are used to achieve exponential amplification of the target sequence. Thus arrangement is useful where all of the target nucleic acids comprise a variable region that is sought to be genotyped and where the variable region is flanked on both sides by conserved regions. In another aspect, multiple pairs of target-specific primers are utilized in a single reaction for amplifying multiple targets nucleic acids simultaneously.
Generally, suitable target-specific primer pairs are short synthetic oligonucleotides, for example having a length of more than 10 nucleotides and less than 50 nucleotides. Target-specific, oligonucleotide primer design involves various parameters such as string-based alignment scores, melting temperature, primer length and GC content. When designing a target-specific primer, one of the important factors is to choose a sequence within the target fragment that is specific to the nucleic acid molecule to be amplified. Another important factor is to calculate the melting temperature of a target-specific primer for the reaction. The melting temperature of a target-specific primer is determined by the length and GC content of that oligonucleotide. Preferably the melting temperature of a primer is about 10 to 30° C. higher than the temperature at which primer hybridization and target amplification will take place.
“Primer hybridization” refers to binding of an oligonucleotide primer to a region of the single-stranded nucleic acid template under the conditions in which the primer binds only specifically to its complementary sequence on one of the template strands, not other regions in the template. The specificity of hybridization may be influenced by the length of the oligonucleotide primer, the temperature in which the hybridization reaction is performed, the ionic strength, and the pH of the reaction mixture.
Each target-specific primer hybridizes to each end of the target nucleic acid and may be extended in a 3′→5′ direction by a polymerase using the target nucleotide sequence as a template. To achieve specific amplification, a homologous or perfect match target-specific primer is preferred. However, target-specific primers may include sequences at the 5′ end which are non-complementary to the target nucleotide sequence(s). Alternatively, target-specific primers may contain nucleotides or sequences throughout that are not exactly complementary to the target nucleic acid.
The target-specific primers may include any of the deoxyribonucleotide bases A, T, G or C and/or one or more ribonucleotide bases, A, C, U, G and/or one or more modified nucleotide (deoxyribonucleotide or ribonucleotide) wherein the modification does not prevent hybridization of the primer to the nucleic acid or elongation of the target-specific primer or denaturation of double stranded molecules. Target-specific primers may be modified with chemical groups such as phosphorothioates or methylphosphonates or with non nucleotide linkers to enhance their performance or to facilitate the characterization of amplification products.
In general, the temperature of denaturation suitable for permitting specificity of target-specific primer-template recognition and subsequent annealing may occur over a range of temperatures, for example 20° C. to 75° C. A preferred denaturation temperature may be selected according to which helicase is selected for the melting process. Tests to determine optimum temperatures for amplification of a nucleic acid in the presence of a selected helicase can be determined by routine experimentation by varying the temperature of the reaction mixture and comparing amplification products using gel electrophoresis.
In a further aspect, amplification is a sequence-independent amplification. As used herein, “sequence-independent amplification” refers to any amplification that does not amplify a specific sequence. By way of example and not limitation, random primer mixtures or nick-inducing agents may be used to initiate sequence-independent amplification.
As used herein, “random primer mixture” refers to mixtures of short randomly generated oligonucleotide sequences.
As used herein, “nick-initiated polymerase activity” refers to polymerase activity in the absence of exogenous primers, which is initiated by single-strand breaks in the template. Synthesis initiates at the single-strand break in the DNA, rather than at the terminus of an exogenous synthetic primer. With nick-initiated synthesis, removal of primers is unnecessary, reducing cost, handling time and potential for loss or degradation of the product. In addition, nick-initiated synthesis reduces false amplification signals caused by self-extension of primers. The nicks may be introduced at defined locations, by using enzymes that nick at a recognition sequence, or may be introduced randomly in a target polynucleotide. As used herein, “nick-inducing agent” refers to any enzymatic or chemical reagent or physical treatment that introduces breaks in the phosphodiester bond between two adjacent nucleotides in one strand of a double-stranded nucleic acid. Examples of nick-inducing enzymes include Bpu10 I, BstNB I, Alw I, BbvC I, BbvC I, Bsm I, BsrD, and E. coli endonuclease I. In one aspect, at least one nick-inducing enzyme is included as a replacement for a helicase in a reaction mixture. In another aspect, at least one nick-inducing enzyme is added to a reaction mixture in addition to at least one helicase.
In one aspect, the amplification is an isothermic amplification. In another aspect, the isothermic amplification is a Whole Genome Amplification (“WGA”). WGA is an isothermal process that uses non-specific primers to generate amplicons using the target nucleic acid sequence as a template. As multiple random primers are used, substantially the entire molecule comprising the target nucleic acid can be amplified using WGA. For example, Phi 29 DNA polymerase can be used in combination with non-specific primers to amplify target nucleic acid sequences. The polymerase can move along the target nucleic acid sequence displacing the complementary strand. The displaced strand becomes a template for replication allowing high yields of high-molecular weight DNA to be generated. In one aspect, the WGA reaction is modified to include at least one helicase, at least one nick-inducing agent, or both.
In a further aspect, the amplicons generated by the amplification step can be fragmented after amplification.
IV. Genotyping
A. Capture
After the at least one target nucleic acid is amplified, it is contacted with at least one polynucleotide probe under a condition sufficient for the one or more polynucleotide capture probes to hybridize to the at least one target nucleic acid. The at least one polynucleotide capture probe can be full length, truncated, or synthetic DNA or full length, truncated, or synthetic RNA.
Where a plurality of target nucleic acids are desired to be genotyped, at least one polynucleotide capture probe specific for each target nucleic acid should be provided. In an aspect, a plurality of polynucleotide probes is used to purify the target nucleic acid. The plurality of polynucleotide probes may consist of only a single nucleic acid probe specific for each target nucleic acid or may consist of a plurality of nucleic acid probes specific for each target nucleic acid. In one aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotide capture probes specific for each single target nucleic acid may be provided. In another aspect, each polynucleotide capture probe is selected such that it is specific only for one target nucleic acid and does not cross-react with any other target nucleic acid in stringent conditions. In yet another aspect, at least two polynucleotide capture probes are provided for each target nucleic acid, wherein each polynucleotide capture probe hybridizes to a distinct region of the target nucleic acid. By way of example, where the target nucleic acids comprise HPV nucleic acids, at least one polynucleotide capture probe may be selected to hybridize to each of the E6/E7 and L1 regions of each HPV nucleic acid to be tested.
The polynucleotide capture probes can be adapted to be immobilized to a second solid support. In one aspect, the polynucleotide capture probes are immobilized to the second solid support before they are hybridized to the at least one target nucleic acid. Supports include but are not limited to beads; magnetic beads, including paramagnetic, diamagnetic, ferromagnetic, ferromagnetic, and diamagnetic beads; columns; plates; filter paper; polydimethylsiloxane (PDMS); dipsticks; tubes; dishes; mica chips.
In a further aspect, the second solid support comprises beads. In one aspect, a plurality of beads are provided, wherein each bead of the plurality immobilizes only polynucleotide capture probes specific for only a single target nucleic acid, such that each bead will specifically immobilize only a single target nucleic acid. In a further aspect, each bead of the plurality bears a detectable label, wherein the detectable label corresponds to the genotype of the target nucleic acid for which the bead is specific.
In one aspect, polystyrene microspheres are provided as the second solid support. Polystyrene microspheres can be filled with various dyes, permitting each individual microsphere to be detectably labeled. In one aspect, polystyrene microspheres marketed under the brand name Luminex® are used. Luminex® microspheres are internally dyed with various concentrations of red and infrared fluorophores, such that 100 different spectral signatures can be generated. In this way, microspheres specific for 100 different target nucleic acids may be generated by immobilizing polynucleotide capture probes specific for a single target nucleic acid to a set of beads with a single identifiable label.
In an aspect, the polynucleotide capture probe is capable of hybridizing or binding to nucleic acids at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, at least 99%, or 100% identical to nucleic acids associated with HPV, genetic variants of HPV, HPV DNA of a HR-HPV type, or HPV RNA of a HR-HPV type, or any one of one of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82; or any one of LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91. In another aspect, the immobilization probe is complementary to HPV, genetic variants of HPV, HPV DNA of a HR-HPV type, HPV RNA of any one of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 or any one of LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91.
In another aspect, a plurality of polynucleotide capture probes is provided, the plurality being selected to hybridize to each of a set of target nucleic acids. In one aspect, the plurality of polynucleotide capture probes is capable of hybridizing to each nucleic acid of a set of target nucleic acids consisting of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 nucleic acids, or any subset thereof. In one aspect, the plurality of polynucleotide capture probes is capable of hybridizing to each nucleic acid of a set of target nucleic acids consisting of LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91, or any subset thereof. In one aspect, the plurality of polynucleotide capture probes is capable of hybridizing to each nucleic acid of a set of target nucleic acids consisting HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82, or any subset thereof; and LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91, or any subset thereof. In a further aspect, a plurality of second solid supports is provided, consisting of solid supports specific for each nucleic acid of a set of target nucleic acids consisting of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82, or any subset thereof; and LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91, or any subset thereof.
Hybridization conditions suitable for the particular probes and diluents described herein are employed. For example, the probes and sample nucleic acids can be incubated for a hybridization time, preferably at least about 5 to about 30 minutes, about 5 to about 20 minutes, or from about 7 to about 15 minutes, or about 10 minutes, as well as any number within the recited ranges sufficient to allow the one or more polynucleotide probes to anneal to a corresponding complementary nucleic acid sequence. The hybridization condition can include a hybridization temperature of at least about 65° C., about 68.5° C., and about 67° C. to about 70° C., as well as any number within the recited ranges. For a given at least one target nucleic acid and a given probe, one of ordinary skill in the art can readily determine desired hybridization conditions by routine experimentation. One of ordinary skill in the art will further appreciate that the time and temperature of hybridization must be optimized, one with respect to the other. Thus, higher hybridization temperatures may be carried out for shorter periods of time and vice versa. Without being limited, stringent hybridization conditions may be controlled by increasing the temperature, increasing the ionic conditions to above 0.5M (using, for example, NaCl), or reducing the concentration of PAA. As a non-limiting example, stringent hybridization conditions may include performing a hybridization reaction at elevated temperatures, such as of at least about 65° C., at least about 68.5° C., between about 67° C. to about 70° C., and between about 69° C. to about 70° C. Stringent hybridization conditions may also include elevated temperatures, such as of at least about 65° C., at least about 68.5° C., and between about 67° C. to about 70° C.
B. Detection
In one aspect, the immobilization probe forms a DNA:RNA hybrid with the amplicon when hybridized thereto. In such a circumstance, detection may be performed using a by providing a second antibody that is also specific for double-stranded DNA:RNA hybrids. The second antibody may be detectably labeled, either directly or indirectly, and may be a monoclonal or polyclonal antibody.
Alternatively, the amplicon may be further hybridized to at least one polynucleotide detection probe specific for the at least one target nucleic acid. The detection probe may be DNA, RNA, synRNA, or PNA and may optionally be detectably labeled. In one aspect, the detectable label is biotin, which may be detected by conjugating the biotin with a streptavidin labeled with a fluorophore, including phycoerythrin.
In one aspect, each detection probe is specific for only a single target nucleic acid and does not cross-react with another target nucleic acid.
In another aspect, a plurality of polynucleotide detection probes is used. The plurality of polynucleotide probes may consist of only a single nucleic acid probe specific for each target nucleic acid or may consist of a plurality of nucleic acid probes specific for each target nucleic acid. In one aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotide capture probes may be provided that are specific for each single target nucleic acid. In another aspect, each polynucleotide capture probe is selected such that it is specific only for one target nucleic acid and does not cross-react with any target nucleic acid in stringent conditions. In yet another aspect, at least two polynucleotide capture probes are provided for each target nucleic acid, wherein each polynucleotide capture probe hybridizes to a distinct region of the target nucleic acid. By way of example, where the target nucleic acids comprise HPV nucleic acids, at least one polynucleotide may be chosen for each of the E6/E7 and L1 regions of the HPV nucleic acid.
In another aspect, a single polynucleotide detection probe is provided that is capable of hybridizing with all of the target nucleic acids under stringent conditions.
In an aspect, the polynucleotide detection probe is capable of hybridizing or binding to nucleic acids at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, at least 99%, or 100% identical to nucleic acids associated with HPV, genetic variants of HPV, HPV DNA of a HR-HPV type, or HPV RNA of a HR-HPV type of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, or 82, or LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, or 91. In another aspect, the detection probe is complementary to HPV, genetic variants of HPV, HPV DNA of a HR-HPV type, HPV RNA of a HR-HPV type, or any one of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82; or any one of LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91.
In another aspect, a plurality of polynucleotide detection probes is provided, the plurality being selected to hybridize to each of a set of target nucleic acids. In one aspect, the plurality of polynucleotide detection probes is capable of hybridizing to each nucleic acid of a set of target nucleic acids consisting of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 nucleic acids, or any subset thereof. In one aspect, the plurality of polynucleotide detection probes is capable of hybridizing to each nucleic acid of a set of target nucleic acids consisting of LR-HPV types 6, 11, 40, 43, 53, 61, 67, 69, 70, 71, 72, 81, and 83. In one aspect, the plurality of polynucleotide detection probes is capable of hybridizing to each nucleic acid of a set of target nucleic acids consisting of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82, or any subset thereof; and LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91, or any subset thereof.
In another aspect, each polynucleotide detection probe bears the same detectable label.
In another aspect, the polynucleotide detection probes are used to generate double stranded nucleic acid hybrids, which can then be detected by providing a second antibody that is also specific for double-stranded nucleic acids hybrids. The second antibody may be detectably labeled, either directly or indirectly, and may be a monoclonal or polyclonal antibody. In an aspect, the second antibody is monoclonal. In another aspect, the second antibody is directly labeled with a detectable marker and is monoclonal. The second antibody is used to detect the presence of double-stranded nucleic acid hybrids. In one aspect, the second antibody has a label that must react with a substrate to provide a signal that can be detected. The second antibody may be dissolved in a suitable buffer. In one aspect the buffer comprises 100 mM TrisHCl, pH 7.4, 0.5 M NaCl, 0.1 mM ZnCl2, 1.0 mM MgCl2, 0.25% Tween 20, 0.2 mg/ml RNase A, 4% hydroxypropyl-b-cyclodextrin (cyclodextrin), 30% bead dilution buffer as discussed previously, 0.05% goat IgG, 0.05% sodium azide.
It will be understood by those skilled in the art that any detectable label such as, but not limited to, an enzyme, radioactive molecule, fluorescent molecule, or metal particle such as gold particle can be used. In certain aspects, the detectable label may be alkaline phosphatase. Methods of conjugating a label to an antibody are known. For example, an antibody can be reduced with dithiothreitol (DTT) to yield monovalent antibody fragments. The reduced antibody can then be directly conjugated to maleinated alkaline phosphatase by the methods of Ishikawa et al., J. Immunoassay 4:209-237 (1983) and Means et al., Chem. 1: 2-12 (1990), the contents of each of which are incorporated herein by reference in its entirety, and the resulting conjugate can be purified by HPLC. The conjugate may also be purified using any type of size-exclusion chromatography. One benefit of purification is that the conjugates of one protein to one antibody can be separated from those conjugates with other ratios of protein to antibody.
In another aspect, the double-stranded nucleic acid hybrids can be detected with a second anti-hybrid antibody that is not directly labeled. For example, the second antibody can be a mouse immunoglobulin that is detected by a labeled goat anti-mouse antibody.
The label present on the labeled solid support may be used to identify the particular genotype of the target nucleic acid. The label on the detection probe or detection antibody may convey information about the quantity of each target nucleic acid purified and may, in addition, convey additional information about the genotype of the target nucleic acids.
Methods for detecting various labels are known in the art. For example, colorimetry, radioactive, surface plasmon resonance, or chemiluminescence methods are described by e.g., Coutlee et al., J. Clin. Microbiol. 27:1002-1007 (1989), the contents of which are incorporated herein by reference in its entirety. For example, a bound alkaline phosphatase conjugate can be detected by chemiluminescence with a reagent such as a LUMI-PHOS 530 reagent (Lumigen, Detroit, Mich.) or DR2 (Applied Biosystems, Foster City, Calif.) using a detector such as an E/LUMINA luminometer (Source Scientific Systems, Inc., Garden Grove, Calif.), an OPTOCOMP I Luminometer (MGM Instruments, Hamden, Conn.), or the like, such as a Veritas Microplate Luminometer by Turner Biosystems. Multiple detection techniques can also be used in sequence or in parallel. For example, the conjugate may be detected by chemiluminescence and fluorescence. In another aspect, the conjugate can be detected by chemiluminescence.
Detectors using different detection techniques for the conjugate may be reversible or irreversibly attached, for example in a modular fashion, to a machine that is capable of performing the method for determining the presence of at least one target nucleic acid in a sample.
All probes used herein (including hybrid, capture, and detection probes) may be short synthetic RNA probes that specifically bind only to the at least one target nucleic acid. Examples are described in U.S. Patent Application Publication No. US 2009-0298187 A1, the contents of which are incorporated herein by reference in its entirety.
The present disclosure also provides for assay compositions, probes, and conditions wherein cross-reactivity between HR-HPV probe sets and LR-HPV types is dramatically reduced when compared to the standard FDA approved HPV assay and probe set. In one aspect, the HPV high-risk probe set is selected from the group consisting of HPV high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 or LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91. Using the present assay with these HR-HPV probes, cross-reactivity between LR-HPV types and HR-HPV probes is reduced. See, for example, U.S. Patent Application Publication No. US 2009-0298187 A1.
The present disclosure also provides methods and assays for detecting cancer, for example cervical cancer, by detecting the presence of a at least one target nucleic acid, such as HPV, in a sample.
It will be understood to those skilled in the art that the present invention can be carried out on a number of platforms including, but not limited to, tubes, dipsticks, microarrays, microplates, 384 well plates, other microtiter plates and microfluidic systems. It will be understood to those skilled in the art that the present, as relevant to developing countries, can utilize low technology methods such as dropper bottles, rubber bulbs, Pasteur pipettes, or squirt bottles for steps involving movement of liquid. These devices deliver relatively precise volumes within the approximate ranges that are needed for the assay. In an aspect, the methods of the disclosure do not include automatic pipettors or other battery powered or energy powered pipetting devices.
In an aspect, 10 copies or fewer of the at least one target nucleic acid can be purified and genotyped by the methods described herein in a volume of about 1 ml-20 ml of collection media in a time period of about 30 minutes to about 3 hours. In other aspects, 10 copies or fewer, 25 copies or fewer, or 50 copies or fewer of a at least one target nucleic acid can be detected by the methods described herein in a volume of about 1 ml of collection media in a time period of about 30 minutes to about 1 hour. In an aspect, the at least one target nucleic acid is at least one HPV nucleic acid selected from the group consisting of HPV high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 and LR-HPV types 6, 11, 40, 43, 53, 61, 67, 69, 70, 71, 72, 81, and 83.
V. Kit
Also provided is a kit for the detection of a at least one target nucleic acid in a sample, the kit comprising, consisting of, or consisting essentially of:

    
    
        i. a collection medium;
        ii. a denaturation reagent;
        iii. at least one polynucleotide hybrid probe;
        iv. a bead coated with a first anti-hybrid antibody;
        v. a polymerase;
        vi. a helicase;
        vii. a plurality of beads coated with immobilization probes, wherein each bead is coated with an immobilization probe specific for a single target nucleic acid and wherein each bead is detectably labeled;
        viii. a detection probe, wherein the detection probe may optionally be labeled, wherein the optional label is selected from the group consisting of: biotin, a His tag, protein G, a fluorophore; and
        ix. optionally comprising a detection reagent selected from the group consisting of: a compound that reacts with a detectable label on the detection probe, including streptavidin:HRP complexes; a second anti-hybrid antibodies bearing a second detectable label; and
        x. a wash buffer.
    
    


The collection medium, denaturation reagent, beads, first and second antibodies, polynucleotide probes, detection reagents, and wash buffers have been previously described.
In an aspect, a plurality of hybrid probes, a plurality of capture probes, and a plurality of detection probes are provided with the kit, wherein the plurality of each probe is specific for a set of target nucleic acids consisting of HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82, or any subset thereof; and LR-HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91, or any subset thereof.
The kit may also include instructions for describing procedures associated with the disclosed methods and assays. The kit may also include a means for transcribing patient information. In an aspect, the means includes paper, a computer, or a device capable of transmitting patient information. The kit can include all the necessary components to complete the methods at the same location where the patient sample is taken.
In an aspect, the kit may include color coded reagents associated with the detection assay. The reagent vials are color coded for ease of use and can be included in a kit. The reagent bottles may also be identified by symbols, letters, or other known identifiers.
As the individual components of the kit come together in an easy to use platform, one advantage of the kit described herein is that it provides for immediate testing of samples. This allows for rapid determination of patient results.
In an aspect, methods of the disclosure can include the collection, processing, and performing the purifying step on patient samples in the field. In one aspect, after the samples are collected, some of the method steps are conducted at the same location where the patient samples are collected. The location may be a village, clinic, laboratory, or communal area where individuals receive medical checkups and evaluations. The location may be permanent or temporary. In an aspect, the nucleic acid is detected at a location, such as a laboratory or clinic, which is different from where the samples are taken. In an aspect, the kit is designed for use in a developing country or geographical areas where access to medical care is not readily available.
This method is further compatible with STM and PC samples.
The following examples are illustrative only and are not intended to limit the disclosure in any way.
EXAMPLES
Among the many possible target nucleic acid sequences which may be purified, detected, and/or characterized by the above-described method, HPV nucleic acid sequences provide an excellent illustrative example.
Members of the HPV family are associated with a number of different disorders and/or infections, including common warts, genital warts, and cancers of the head, neck, throat, penis, anus, cervix, vulva, and vagina. Over 100 types of HPV viruses have been described, 56 of which have been associated with mucosal and/or cutaneous lesions to date. These 56 mucosal and/or cutaneous lesion-associated HPV types are typically segregated into “high-risk” and “low-risk” groups. HR-HPV are those that are associated with malignant lesions and may include, for example, HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82. LR-HPV are associated benign lesions and may include, for example, HPV types 2, 3, 6, 7, 10, 11, 13, 27, 28, 30, 32, 40, 42, 43, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74, 81, 83, 84, 85, 86, 87, 89, 90, and 91.
However, prior to the development of the above-described method, there were no tests available which were able to genotype all known HR- and LR-HPV types. Other molecular diagnostic methods of detecting HPV are limited in their multiplexing capability, decreasing the usefulness of those methods for genotyping. Chemiluminescent methods, such as Hybrid Capture 2, are sensitive and reliable, but have a homogeneous output, necessitating separate wells for each genotype to be detected. PCR and PCR-like tests rely on the use of consensus primer (e.g. GP5+/6+, MY09/MY11, etc.). These tests inherently have different efficiencies for different targets, resulting in amplification bias, making detection of multiple HPV infection difficult and unreliable. Additionally, cross-talk among fluorophores, competition during amplication, and increasing problems with primer-dimers negatively affect and limit the number of targets that can be amplified and detected simultaneuously. Moreover, another limitation is the relatively small size of the targeted amplicon region which in turn results in assays that are sensitive to deletions, mutations, or insertions. For example, if this targeted region is deleted during viral integration into the host genome (as has been shown with the L1 region), the infection will be missed. Additionally, false negatives may occur when there are mutations or deletions in the region targeted by the detection probe.
In the following examples, the above described methods were used to purify, detect, and characterize 26 HPV types, including all currently known HR-HPVs.
Example 1
Assay Design
The following examples all utilize the same general assay design. First, HPV nucleic acids are isolated from the sample through the use of hybrid capture. Then, the isolated HPV nucleic acids are amplified using whole genome amplification. The amplified HPV nucleic acids are then segregated according to HPV serotype using capture probes specific for each individual HPV serotype immobilized to a uniquely labeled bead. A biotinylated detection probe is then hybridized to the segregated HPV nucleic acids and a streptavidin/phycoerythrin (SA-PE) conjugate is bound to the detection probe to generate a signal. Both the unique label of the bead and the signal generated by the SA-PE are measured using flow cytometry. The bead label is used to indicate the genotype bound to the bead, while the SA-PE signal is used to indicate the presence or absence of bound HPV nucleic acid to each bead.
A. Purification Probe Preparation.
A purification probe set was designed so as to be able to isolate nucleic acids of all of the HPV serotypes being. Although in principle the probes could be any length, short 25-mer probes were selected to provide flexibility of probe design and ease of manufacture.
The purification probe set was prepared using two basic criteria. First, the probes were selected such that they were dispersed throughout the target genome so as to capture all regions of the genome. Second, multiple probes were clustered around specific regions to ensure that each region being tested was purified.
To minimize the total number of required probes, the probes were designed so that a single probe could be used as a consensus probe for two or more HPV types. To discover the consensus sequences, alignments of the HPV sequences being tested were performed in a family-wise fashion. For example, all members of the A9 (HPV 16) family, comprising HPVs 16, 31, 33, 35, 52, 58, & 67, or the A7 (HPV18) family, comprising HPV18, HPV39, HPV45, HPV59, HPV68, HPV70, and HPV85, were aligned by ClustalW and the consensus sequence was analyzed for the presence of a contiguous 25-mer sequence. Such a sequence would then be chosen as a probe candidate. Based on the phylogenetic tree constructed during the analysis, the two most closely related sequences were then aligned with one another and the search for consensus sequences was repeated. Thus, the probes enumerated in Table 1 (below) were designed.







TABLE 1







PURIFICATION PROBE SEQUENCES FOR 27


TYPES OF HPV











SEQ





ID





NO.
Name
Sequence







  1
HPV16-E6E7-1
AACCGAAACCGGUUAGUAUAAAAGC



 



  2
HPV16-E6E7-2
UUAGAAUGUGUGUACUGCAAGCAAC



 



  3
HPV16-E6E7-3
GGUAUAUGACUUUGCUUUUCGGGAU



 



  4
HPV16-E6E7-4
AGUAUAUAGAGAUGGGAAUCCAUAU



 



  5
HPV16-E6E7-5
ACAACAAACCGUUGUGUGAUUUGUU



 



  6
HPV16-E6E7-6
UUAGGUGUAUUAACUGUCAAAAGCC



 



  7
HPV16-E6E7-7
GAUUCCAUAAUAUAAGGGGUCGGUG



 



  8
HPV16-L1-1
AUGUUGCACGCACAAACAUAUAUUA



 



  9
HPV16-L1-2
GUUCCUAAAGUAUCAGGAUUACAAU



 



 10
HPV16-L1-3
UCCCUAUUUUCCUAUUAAAAAACCU



 



 11
HPV16-L1-4
GUUUGGGCCUGUGUAGGUGUUGAGG



 



 12
HPV16-L1-5
GUCAGCCAUUAGGUGUGGGCAUUAG



 



 13
HPV16-L1-6
UGUGUUUAAUUGGUUGCAAACCACC



 



 14
HPV16-L1-7
GGUGAUUGUCCACCAUUAGAGUUAA



 



 15
HPV16-L1-8
CAGUUAUUCAGGAUGGUGAUAUGGU



 



 16
HPV16-A9-1
GAAAUCCUUUUUCUCAAGGACGUGG



 



 17
HPV16-A9-2
CAAUGUGUAGACAUUAUAAACGAGC



 



 18
HPV16-A9-3
UUAGACAUUUAUUUAAUAGGGCUGG



 



 19
HPV16-A9-5
CAUAUGAUAAUCCUGCAUAUGAAGG



 



 20
HPV16-A9-6
AAUAUGAUUUACAGUUUAUUUUUCA



 



 21
HPV16-A9-7
AUCCUUUAUUAAAUAAAUUGGAUGA



 



 22
HPV16/35-A9-8
CUAUUAGUACACAUAAUUAUGAAGA



 



 23
HPV31/35-A9-1
GGUACAAUGGGCAUAUGACAAUGAU



 



 24
HPV31/35-A9-2
GACAAACAGUAUUACAGCAUAGUUU



 



 25
HPV31/35-A9-3
GAUGGUAUAGAUAUAGUGUGUAUGG



 



 26
HPV31/35-A9-4
GAUUAAAUUUGCACGAGGAAAGAGG



 



 27
HPV31-E6E7-1
ACGAUGAACUAAGAUUGAAUUGUGU



 



 28
HPV31-E6E7-2
ACAGAGGUAUUAGAUUUUGCAUUUA



 



 29
HPV31-E6E7-3
UUAAUUAGGUGUAUAACGUGUCAAA



 



 30
HPV31-E6E7-4
AGAAGAAAAACAAAGACAUUUGGAU



 



 31
HPV31-E6E7-5
AGGAAGGUGGACAGGACGUUGCAUA



 



 32
HPV31-L1-1
GAACCAACAUAUAUUAUCACGCAGG



 



 33
HPV31-L1-2
AUCCAUAUUAUUCCAUACCUAAAUC



 



 34
HPV31-L1-3
UCAGGAUUACAAUAUAGGGUAUUUA



 



 35
HPV31-L1-4
GACACUGAAAACUCUAAUAGAUAUG



 



 36
HPV31-L1-5
AAUGUAUAUCAAUGGAUUAUAAACA



 



 37
HPV31-L1-6
CUAUUGGAGAGCAUUGGGGUAAAGG



 



 38
HPV31-L1-7
GGUGAUUGUCCUCCAUUAGAAUUAA



 



 39
HPV31-E6E7-6
UAGUAUAUAGGGACGACACACCACA



 



 40
HPV31-E6E7-7
CUGAAACCCAAGUGUAAACAUGCGU



 



 41
HPV35-E6E7-1
GCUAUGAUUUGUGUAUAGUAUAUAG



 



 42
HPV35-E6E7-2
UCCAGUUGAAAAGCAAAGACAUUUA



 



 43
HPV35-E6E7-3
UUGUAAAUGUGAGGCGACACUACGU



 



 44
HPV35-E6E7-4
AAGAUUUAUUAAUGGGCACAUUUGG



 



 45
HPV35-L1-1
GCACAAACAUCUACUAUCAUGCAGG



 



 46
HPV35-L1-2
CAAUACAGAGUAUUUAGAGUAAAAU



 



 47
HPV35-L1-3
CUAAUAAGUUUGGAUUUCCAGACAC



 



 48
HPV35-L1-4
UGGUUUGGGCCUGUACAGGAGUUGA



 



 49
HPV35-L1-5
GUACAGAUAACAGGGAAUGCAUUUC



 



 50
HPV67-E6E7-1
UAACCGAAAACGGUUUGACCGAAAA



 



 51
HPV67-E6E7-2
CGAAAAACCACGCAACCUGCACGAA



 



 52
HPV67-E6E7-3
CUUUGGAAACCACGGUGCAUGAAAU



 



 53
HPV67-E6E7-4
CUUUGGACAGAAACGAGGUAUAUGA



 



 54
HPV67-E6E7-5
UCUGUGAGUGCACUUUGCGUUUGUG



 



 55
HPV67-E6E7-6
AAUCCAGCAGAUGCUUAUGAACACA



 



 56
HPV67-L1-1
UAUUGAAAUAGGGCGAGGACAGCCU



 



 57
HPV67-L1-2
CUGAUAAUAGGGAAUGCUUGUCUAU



 



 58
HPV67-L1-3
UUUGGAACUUAUGAAUACUGUUAUU



 



 59
HPV67-L1-4
GAGCAGGUAAAUUAGGGGAGGAUGU



 



 60
HPV67-L1-5
GCAAACACUUCUGCACUGCAAACCU



 



 61
HPV67-L1-6
GCUAAACCUAAACUAAAACGUUCUU



 



 62
HPV67-L1-7
CAAAACGUAAAAAGGUUAAAAGGUA



 



 63
HPV52-E6E7-1
GUGUAGCUAACGCACGGCCAUGUUU



 



 64
HPV52-E6E7-2
UGCACGAAUUGUGUGAGGUGCUGGA



 



 65
HPV52-E6E7-3
UACAACGAAGAGAGGUAUACAAGUU



 



 66
HPV52-E6E7-4
ACGAAUAGUAUAUAGAGACAAUAAU



 



 67
HPV52-E6E7-5
GGCAUUAUCAAUAUUCACUGUAUGG



 



 68
HPV52-E6E7-6
CUAUGAGCAAUUAGGUGACAGCUCA



 



 69
HPV52-E6E7-7
GCCAGAUGGACAAGCAGAACAAGCC



 



 70
HPV52-L1-1
UAACAGUAGGACAUCCCUAUUUUUC



 



 71
HPV52-L1-2
AAAAAAGUUUUAGUUCCCAAGGUGU



 



 72
HPV52-L1-3
UUUGAUGAUACUGAAACCAGUAACA



 



 73
HPV52-L1-4
AGGGAAUGUUUAUCUAUGGAUUAUA



 



 74
HPV52-L1-5
UGCAAACCUCCUAUAGGUGAACAUU



 



 75
HPV52-L1-6
AGGAUGGGGACAUGGUAGAUACAGG



 



 76
HPV33/58-A9-1
UAGAAGACAGCGGAUAUGGCAAUAC



 



 77
HPV33/58-A9-2
GCUGUACAGAUUGGUGUAUAACAGG



 



 78
HPV33/58-A9-3
AUUGGUUUAGAACAGCAAUGUCAAA



 



 79
HPV33/58-A9-4
UCAUAUUUUGGAAUGAGUUUAAUAC



 



 80
HPV33/58-A9-5
UUUUGGUUGCAGCCAUUAUCAGAUG



 



 81
HPV33/58-A9-6
AAUAGAGGAAGAGGACAAGGAAAAC



 



 82
HPV33/58-A9-7
AUGGAGGAAAUAUCAGCACGUUUAA



 



 83
HPV33/58-A9-8
CAUCACAAAUUGAACAUUGGAAACU



 



 84
HPV33/58-A9-9
GGACAUUGCAACAAACAAGCUUAGA



 



 85
HPV33/58-A9-10
AUUUUAAAUAUUUUAAAGAGGAUGC



 



 86
HPV33/58-A9-11
AAUAUCCACUACUGAAACUGCUGAC



 



 87
HPV33/58-A9-12
CAAAUAGUUUAAAAUGUUUAAGAUA



 



 88
HPV33/58-A9-13
UGAUGUGUAUUAAUUUUCAUGCACA



 



 89
HPV33/58-A9-14
UCUACAAGGCGCAAGCGUGCAUCUG



 



 90
HPV33/58-A9-15
GAAAACAUACCAAUGGAUACCUUUG



 



 91
HPV33/58-A9-16
GCCCUGUGGCACGCCUUGGUUUAUA



 



 92
HPV33/58-A9-17
CAGAUGUCCGUGUGGCGGCCUAGUG



 



 93
HPV33/58-A9-18
ACAUUGCAGGCUAAUAAAAGUGAUG



 



 94
HPV33/58-A9-19
CAGUACAUGCAAAUAUCCAGAUUAU



 



 95
HPV52/67-A9-1
GAGAAAUGGUGCAAUGGGCAUAUGA



 



 96
HPV52/67-A9-2
UUUUUAAAAGGUAUACCUAAAAAAA



 



 97
HPV52/67-A9-3
UAACAGUGCAAUACGAUAAUGAUAA



 



 98
HPV52/67-A9-4
CAGAUGUCCGUGUGGCGGCCUAGUG



 



 99
HPV52/67-A9-5
AGGCCACUGUGUACCUGCCUCCUGU



 



100
HPV52/67-A9-6
UUAGAGGACUGGCAAUUUGGCCUUA



 



101
HPV52/67-A9-7
CAUGUUUUAAACUGCUUUUAGGCAC



 



102
HPV18/45-A7-1
AUGCUGCAUGCCAUAAAUGUAUAGA



 



103
HPV18/45-A7-2
UUAAAACGAAAGUUUGCAGGAGGCA



 



104
HPV18/45-A7-3
UCAGAUAGUGGCUAUGGCUGUUCUG



 



105
HPV18/45-A7-4
UUAGAAAUUUUAAAAGUGAUAAAAC



 



106
HPV18/45-A7-5
UGUAAAUGGGGAGUAUUAAUAUUAG



 



107
HPV18/45-A7-6
CACCAAAAUUGCGAAGUAGUGUUGC



 



108
HPV18/45-A7-7
AUGCAUUUCCAUUUGAUAAAAAUGG



 



109
HPV18/45-A7-8
GAAAGGACAUGGUCCAGAUUAGAUU



 



110
HPV18/45-A7-9
UUGAUUGUAAUGACUCUAUGUGCAG



 



111
HPV18/45-A7-10
UACCAGUGACGACACGGUAUCCGCU



 



112
HPV18/45-A7-11
GUGGUAACACUACGCCUAUAAUACA



 



113
HPV18/45-A7-12
GUAAUAAAACUGCUUUUAGGCACAU



 



114
HPV18-E6E7-1
AGGAUCCAACACGGCGACCCUACAA



 



115
HPV18-E6E7-2
CUUCACUGCAAGACAUAGAAAUAAC



 



116
HPV18-E6E7-3
AGGUAUUUGAAUUUGCAUUUAAAGA



 



117
HPV18-E6E7-4
GAGGCCAGUGCCAUUCGUGCUGCAA



 



118
HPV18-L1-1
UGGUAAUCCAUAUUUUAGGGUUCCU



 



119
HPV18-L1-2
UUCCUAAGGUUUCUGCAUACCAAUA



 



120
HPV18-L1-3
AUCCUGAAACACAACGUUUAGUGUG



 



121
HPV18-L1-4
AGGACGUUAGGGACAAUGUGUCUGU



 



122
HPV45-E6E7-1
UGACGAUCCAAAGCAACGACCCUAC



 



123
HPV45-E6E7-2
UAGACACCUUAAGGACAAACGAAGA



 



124
HPV45-E6E7-3
GUGUGACGGCAGAAUUGAGCUUACA



 



125
HPV45-E6E7-4
UACAGCAGCUGUUUUUGAGCACCUU



 



126
HPV45-L1-1
UGUAGGCAAUCCAUAUUUUAGGGUU



 



127
HPV45-L1-2
UUCCUAAGGUAUCCGCAUAUCAGUA



 



128
HPV45-L1-3
AUAAUCCUGAAACACAACGUUUGGU



 



129
HPV45-L1-4
AGGAUGUUAGGGAUAAUGUGUCAGU



 



130
HPV39/68-A7-1
CCAUACAAAUUGCCAGACCUGUGCA



 



131
HPV39/68-A7-2
UCGGUGUAUGCAACUACAUUAGAAA



 



132
HPV39/68-A7-3
UACAAUGAAAUACAGCCGGUUGACC



 



133
HPV39/68-A7-4
UUGUAUGUCACGAGCAAUUAGGAGA



 



134
HPV39/68-A7-5
GAUGAAAUAGAUGAACCCGACCAUG



 



135
HPV39/68-A7-6
AGCGUGAGACAGCACAGGUACUUUU



 



136
HPV39/68-A7-7
AGUGCUAUAGAUAGUGAAAACCAGG



 



137
HPV39/68-A7-8
GUAAAAGAUUGUGCAACAAUGUGUA



 



138
HPV39/68-A7-9
AAAUUUCCUAAUGCAUUUCCAUUUG



 



139
HPV39/68-A7-10
GAAAAGACUUGGUGCAGAUUAGACU



 



140
HPV39/68-A7-11
GACGAGGAUGAAGGAGACAAUGAUG



 



141
HPV39/68-A7-12
AAGCAUAUCAAGCUAUUGAACUGCA



 



142
HPV39/68-A7-13
CAUUGUCCUGACUCUAUGUGCAGUA



 



143
HPV39/68-A7-14
ACACCAGUACCAACAUUUACAGGCA



 



144
HPV39/68-A7-15
CAGGUUCGUGUUAGUAAUUUUGAUU



 



145
HPV39/68-A7-16
CACCCUUCAUCAUUUGUAACAUUUG



 



146
HPV39/68-A7-17
AUAAUCCUGCUUUUGAGCCUGUUGA



 



147
HPV39/68-A7-18
GAUCCGGAUUUUCUGGACAUUGUUC



 



148
HPV39/68-A7-19
UGCAAAUGUCUGCAGAUGUGUAUGG



 



149
HPV39/68-A7-20
CUAAACACAAACGUAAACGUGUGUC



 



150
HPV59/70-A7-1
GCAACAGAUACAGGUUCAGACUUGG



 



151
HPV59/70-A7-2
AUUUGUGUACAGGCAGAGCGCGAGA



 



152
HPV59/70-A7-3
UUAGUCAUCAUCCUGUCCAGGUGCA



 



153
HPV70-E6E7-1
UAUAAAACCAUGCAAAAGUUGCUUG



 



154
HPV70-E6E7-2
CCUGCAGAACGGCCAUACAAAUUGC



 



155
HPV70-E6E7-3
UGCAUGCCAAAAAUGUAUUAAAUUU



 



156
HPV70-E6E7-4
GACGUAUACGAAGAGAAACACAAGU



 



157
HPV70-E6E7-5
ACAUUGCAAGAGAUUGUUUUAGAUU



 



158
HPV70-E6E7-6
CUACACUGCACUUAGUAGUAGAAGC



 



159
HPV70-E6E7-7
GCAGCUGUUUAUGGAGACACUGUCA



 



160
HPV70-L1-1
GGGUAUCCCUACCUGAUCCUAAUAA



 



161
HPV70-L1-2
UAUAAUCCUGACACACAACGCCUGG



 



162
HPV70-L1-3
CUUCAGAGUUAUAUAUUAAAGGCAC



 



163
HPV70-L1-4
AUGUAUAUUCCCCUUCCCCAAGUGG



 



164
HPV70-L1-5
CACGUAGUACUAAUUUUACAUUGUC



 



165
HPV70-L1-6
CUGUAUAUAGCCCUACAAAGUUUAA



 



166
HPV70-L1-7
UCUAAACACAAACGGAAACGUGUGU



 



167
HPV59-E6E7-1
UUAUAGUGUAUAGAGACUGUACACC



 



168
HPV59-E6E7-2
UUUUAUGCAAGAGUAAGAGAAUUAA



 



169
HPV59-E6E7-3
UAUUAUAGAGAUUCCGUGUAUGGAG



 



170
HPV59-E6E7-4
UGCCUAAAACCUCUAUGUCCAACAG



 



171
HPV59-E6E7-5
ACCACAAAAUUAUGAGGAAGUUGAC



 



172
HPV59-E6E7-6
CUCCGAGAAUGAAAAAGAUGAACCA



 



173
HPV59-L1-1
UGGACAUCCAUAUUUUAAAGUACCU



 



174
HPV59-L1-2
UUCCUAAGGUGUCUGCAUAUCAAUA



 



175
HPV59-L1-3
UGGAUGACACUGAAAACUCUCAUGU



 



176
HPV59-L1-4
GAUAAUGUAUCUGUGGAUUAUAAAC



 



177
HPV59-L1-5
UGAAUCACUAUAUAUUAAAGGUACU



 



178
HPV59-L1-6
UAUUCCCCUUCCCCAAGUGGGUCUG



 



179
HPV59-L1-7
GUGCAGCGCCUGCCCCUACCUCUAC



 



180
HPV59-L1-8
UCUUCCAGAAAAUAGUGUUGUUUGU



 



181
HPV59-na-1
UGUAUUGUUUGCCUGUUUGUAUGUU



 



182
HPV59-na-2
CCGUUUUGUUCAAUCUGCUGCUGUA



 



183
HPV59-na-3
AAGACAGCAACGACAAGCGCGUAGU



 



184
HPV54-E6E7-1
AAGCGGAUGUAGAAAACAGUUAUUU



 



185
HPV54-E6E7-2
ACGGACCAGCCGCGUACUCUAGCUG



 



186
HPV54-E6E7-3
UAUGCAUAGUUUGCAACUUCCUUGU



 



187
HPV54-E6E7-4
GCAGAGAUUUAUGCAUUUCAAUAUA



 



188
HPV54-E6E7-5
GUGGAGACACGGCUUUCCACAUGCU



 



189
HPV54-E6E7-6
AAAUAAAUUAUAGAAGGCAUCGCGA



 



190
HPV54-na-1
UGCAUGGAAAUGUGGCUACAAUUGA



 



191
HPV54-E6E7-7
GUGGAGGUGUGUGUUGUAAGACAGU



 



192
HPV54-E6E7-8
CAUAAGGGUACUGCAGGAACUGCUU



 



193
HPV54-na-2
AAACGAAAGUAUAUAGGCAGUCCGU



 



194
HPV54-na-3
GAGUUUUAUGGACCUAGCACGGUCC



 



195
HPV54-L1-1
UUAUUGGCUGUUGGACAUCCAUAUU



 



196
HPV54-L1-2
UAUUCCUAAAGUAUCAGGAUAUCAA



 



197
HPV54-L1-3
CUAUAGGUGAACACUGGGCUAAAGG



 



198
HPV54-L1-4
GCUGGUGACUGUCCUCCUUUGGAAU



 



199
HPV54-L1-5
GGAUUUUAAAACCCUACAAACCUCA



 



200
HPV54-L1-6
AUUUGUAAAUAUCCUGAUUACCUUA



 



201
HPV54-L1-7
GUAGUACUAACCUAACAUUGUGUGC



 



202
HPV54-L1-8
UUCUGACUUUAGGGAGUAUAUUAGA



 



203
HPV54-na-4
UAUGCUGCAACUCCUAGUGGCUCUA



 



204
HPV70/85-A7-1
UAGAUGACAGUGUAUUUGACCUGUC



 



205
HPV70/85-A7-2
UAUGGGGACAGUAUGUUUUUUUGUU



 



206
HPV70/85-A7-3
GAGGAAUAUGAUUUACAAUUUAUAU



 



207
HPV85-E6E7-1
CUACCCGACCCUACAAACUACCAGA



 



208
HPV85-E6E7-2
AAGAUAUAGAAAUAAGCUGUGUAUA



 



209
HPV85-E6E7-3
AUAGCGACUCUGUGUAUGGGGAAAC



 



210
HPV85-E6E7-4
AUGAUAUAUUAAUAAGGUGUUUACG



 



211
HPV85-E6E7-5
AUAUAAUGAAGUGCAAGAGGUUGAC



 



212
HPV85-E6E7-6
AGGAAGAAAUAGAUGAACCAGAUAA



 



213
HPV85-L1-1
AUGACACAGAAAAUUCCCAUGUUGC



 



214
HPV85-L1-2
GAUAAUGUGUCAGUGGAUUAUAAAC



 



215
HPV85-L1-3
GGGAACAUUGGGCUAAGGGUACUGC



 



216
HPV85-L1-4
UGUCCUCCAUUAGAACUAGUAAAUA



 



217
HPV85-L1-5
GAAACUUAUAUAUAAAAGGUACUAA



 



218
HPV85-L1-6
UAUUCUCCAUCACCUAGUGGGUCUA



 



219
HPV85-L1-7
CAUCUGCCAUUACAUGUCAGAAGGA



 



220
HPV85-L1-8
UAUGAAAAAUUAAAGUUUUGGAAUG



 



221
HPV85-na-1
GUUUUUACUUGCUUUAAUUACACUA



 



222
HPV85-na-2
ACAAGAAAUAUCGUUAAAUAGCUAU



 



223
HPV85-na-3
CAAGGGAGCAUGGUCUUAAAACAAU



 



224
HPV26/69-E6E7-1
GCAGGUACAGUGUGUAUAUUGCAAG



 



225
HPV26/69-E6E7-2
GUGCCGCAACCCGAAAUUGACCUAC



 



226
HPV26/69-E6E7-3
GGACUAUGAACAAUUUGACAGCUCA



 



227
HPV26/69-E6E7-4
GUAAUAGUAUAGUGCAGCUAGCUGU



 



228
HPV26/69-E6E7-5
GUACAGGGUGGUUUUCAGUAGAAGC



 



229
HPV26/69-E6E7-6
AGAACAGCCCGUUGCAAGACAUAAC



 



230
HPV26/69-E6E7-7
AUACUGAAGUGGAAACUCUUACGCC



 



231
HPV26/69-E6E7-8
AGUGUGUGUAGUCAGGGGGGGUCAA



 



232
HPV26/69-na-1
UGUGGCAGGCUCUGUAGCAGAAAGU



 



233
HPV26/69-na-2
AUUUAUCAAAAAUGGUGCAAUGGGC



 



234
HPV26/69-na-3
GGCAAAAUAUGUAAAAGACUGUGCA



 



235
HPV26/69-na-4
GACAGCAAUGGGAAUCCUGUAUAUG



 



236
HPV26/69-na-5
UGGUCCAGAUUAGAUUUGGAGGAGG



 



237
HPV26/69-na-6
AUCUACCUGGCAUUGGACCAGUAAU



 



238
HPV26/69-na-7
GUUUGUGCUUUGCGUGUGUGUGUGU



 



239
HPV26/69-L1-1
CCUUUGAUAAUCCUGCAUAUGAACC



 



240
HPV26/69-L1-2
GUACUAGUGACAGCAAGGUAUAUCU



 



241
HPV26/69-L1-3
GAAACAGCAUGUUUUUUUUUCUUCG



 



242
HPV26/69-L1-4
ACAACACAUCCUGCCUCCUACGCUU



 



243
HPV26/69-L1-5
AAUAAAACUGCUGUUAGGCACAUAU



 



244
HPV51/82-E6E7-1
CACUUGGGCCUGAAGAAAAGCAAAA



 



245
HPV51/82-E6E7-2
GUGUAAUAAAGCCAUGCGUGGUAAU



 



246
HPV51/82-E6E7-3
AAAUUGACUUGCAAUGCUACGAGCA



 



247
HPV51/82-E6E7-4
UGGACAGGCUACGUGUUACAGAAUU



 



248
HPV51/82-E6E7-5
AGCAGCCCAUUAGGAGACAUUACAA



 



249
HPV51/82-E6E7-6
GAUUACUGGACAGUUAUCCGGACAG



 



250
HPV51/82-E6E7-7
UGUGGAAGCAACGUUGCAGGUAGAU



 



251
HPV51/82-na-1
ACAGCCACUAGAGGAUGCUAAAAUA



 



252
HPV51/82-na-2
GUCCAGAUUAGAUUUGGAGGAGGAA



 



253
HPV51/82-na-3
UGCCAGGAGAAAAUACUAGACUGUU



 



254
HPV51/82-na-4
UCAACCUGGCAUUGGACCAGUAAUA



 



255
HPV51/82-na-5
ACAAGCCAAUAUGUGCUGCUAAUUG



 



256
HPV51/82-na-6
UGUGUGUGUGUCUUGUGUUGUGUUG



 



257
HPV51/82-na-7
ACAUGCAAAGCUGCUGGUACAUGUC



 



258
HPV51/82-na-8
UGGAGUGGGUUGGGUAUAUUUUUGG



 



259
HPV51/82-L1-1
GAACUUGAAAUGCAGCCUUUACUUU



 



260
HPV51/82-L1-2
UGUCUUCAUCUUAUGCAAAUGUUAC



 



261
HPV51/82-L1-3
UGGGGAUUACUAUUUGUGGCCCUAU



 



262
HPV51/82-L1-4
AAACGCCGUAAACGUAUACCCUAUU



 



263
HPV51/82-L1-5
UCUUCCUCUUCCUCUUCAGCCAAAC



 



264
HPV30/53-E6E7-1
CCGAAAACGGUACAUAUAAAAGCAC



 



265
HPV30/53-E6E7-2
GACACCAGAGGAAAAACAGUUACAC



 



266
HPV30/53-E6E7-3
AUGAGCAAUUGAACAGCUCAGAGGA



 



267
HPV30/53-E6E7-4
CAAUGGCGUCACCUGAAGGUACAGA



 



268
HPV30/53-E6E7-5
UAAAACGAAAGUAUUUAGGCAGUCC



 



269
HPV30/53-na-1
CAGCGGGUAUGGCAAUACUUUGGAA



 



270
HPV30/53-na-2
ACACAGUCACUUUUGGUUACAACCG



 



271
HPV30/53-na-3
GAAAGGACAUGGUCCAGAUUAGAUU



 



272
HPV30/53-na-4
CGUGCCAGGAGAAAAUUCUAGACUG



 



273
HPV30/53-na-5
UACAAGUGUGUAAAGCAAAGGCAUG



 



274
HPV30/53-na-6
UAAAGGCACAUGGGAAGUGCAUAUG



 



275
HPV30/53-na-7
GUAUUUAUUGUCCCGACUCUGUGUC



 



276
HPV30/53-L1-1
GAAAUACCUAUGCAAACAUUUGCUG



 



277
HPV30/53-L1-2
CACAGACCUGCCUUUACAACACGUA



 



278
HPV30/53-L1-3
GGUGGUGUGCGUUUUAGUAGGCUUG



 



279
HPV30/53-L1-4
AGAAGUGGCAAACAAAUAGGUGCUC



 



280
HPV30/53-L1-5
GAUGGCCUAUAUGAUAUUUAUGCAA



 



281
HPV30/53-L1-6
UUCCCUAUUUUCUUGCAGAUGGCGG



 



282
HPV30/53-L1-7
GCUUAGAGGACAAAUACAGAUAUGU



 



283
HPV30/53-L1-8
UGUAUGACUGUAUGUAUGUGUAAUG



 



284
HPV56/66-E6E7-1
CCGAAAACGGUACAUAUAAAAGGCA



 



285
HPV56/66-E6E7-2
CUCAGAGGAUGAGGAUGAGGAUGAA



 



286
HPV56/66-E6E7-3
GCGGCCACAGCAAGCUAGACAAGCU



 



287
HPV56/66-E6E7-4
GCGUUAACAGUAACGUGCCCACUCU



 



288
HPV56/66-E6E7-5
GCAAGUACAAACAGCACAUGCAGAU



 



289
HPV56/66-na-1
ACAGACGUUGCAAAAACUAAAACGA



 



290
HPV56/66-na-2
AUGAAUAUGUGCCAGUGGAUAAAGC



 



291
HPV56/66-na-3
UGAAGGGGGUGAUUGGAAACCCAUU



 



292
HPV56/66-na-4
GGAUAACGACGAGGACAAAGAAAAC



 



293
HPV56/66-na-5
UGUAAAGCAAAAGCAUGUAGUGCAA



 



294
HPV56/66-na-6
GUCCUGACUCUGUGUCUAGUACCUG



 



295
HPV56/66-na-7
GUAUCCCACAGACCAGGAAAACGAC



 



296
HPV56/66-na-8
GUUUGCGCUUUGCUUUUGUGUUUGU



 



297
HPV56/66-na-9
AUAGGCCUGCAUUUACUACACGUAG



 



298
HPV56/66-L1-1
GAUAUAAGUCCUAUUGCACAGGCUG



 



299
HPV56/66-L1-2
AGGCGCCGUAAACGUAUUCCCUAUU



 



300
HPV56/66-L1-3
CUACCUCCAACACCUGUUUCAAAGG



 



301
HPV56/66-L1-4
UUCUAUGUGGUUUUACUUACGCAGG



 



302
HPV56/66-L1-5
AUAAACCUUAUUGGUUGCAACGUGC



 



303
HPV56/66-L1-6
ACGCGUGGUUGCAUAAACUAAGGUG



 



304
HPV34/73-E6E7-1
UAAUAAGGUGCGGAAAAUGCCAAAA



 



305
HPV34/73-E6E7-2
GACAACUCAGAGGAUGAGGAUGAAA



 



306
HPV34/73-E6E7-3
AGAAGAUGGCUGAUUCAGGUAAUUG



 



307
HPV34/73-E6E7-4
CGGGAUGGUUUAAUGUAGAAGCCAU



 



308
HPV34/73-E6E7-5
UGGGGGAUUUUAUUGAUAAUGCACA



 



309
HPV34/73-E6E7-6
AAUGCAGACAAUGAGGCUAUACGUG



 



310
HPV34/73-E6E7-7
GAUAUGGCAAUACUGAAGUGGAAAC



 



311
HPV34/73-E6E7-8
UAGUGGGUCCAGUAGCAUUUCAAAU



 



312
HPV34/73-na-1
UUUAACAGAGGACGACGACAAGGAA



 



313
HPV34/73-na-2
AAGCCUUGCAGUAUCACGAUCCAAA



 



314
HPV34/73-na-3
UGUUGCAACCUCCUCCACCCUUAGA



 



315
HPV34/73-na-4
GCCUCUGGCAGACUUUUAUUUUCAA



 



316
HPV34/73-na-5
AACAGGUUAAGGUUGUAGACCCUGC



 



317
HPV34/73-na-6
CAGCACAGUGACUUGCAUAAUGCUC



 



318
HPV34/73-na-7
UACUAGAAGUGGCAAACGUAUAGGU



 



319
HPV34/73-L1-1
AAAGGUAUACCUGCCCCCUGUGUCU



 



320
HPV34/73-L1-2
AAAGUUUCAGGUUUGCAAUACAGGG



 



321
HPV34/73-L1-3
CUGUUGUAGAUACUACUAGAAGCAC



 



322
HPV34/73-L1-4
UUUUGGCUUCCUGCAGGCAACUUGG



 



323
HPV34/73-L1-5
UGCACACACAUUUUUUACCCACCCU



 



324
HPV6/11-E6E7-1
GAAAACGGUUCAACCGAAAACGGUU



 



325
HPV6/11-E6E7-2
GACCAGUUGUGCAAGACGUUUAAUC



 



326
HPV6/11-E6E7-3
ACUGCUGGACAACAUGCAUGGAAGA



 



327
HPV6/11-E6E7-4
GACCCUGUAGGGUUACAUUGCUAUG



 



328
HPV6/11-E6E7-5
AGACAGCUCAGAAGAUGAGGUGGAC



 



329
HPV6/11-E6E7-6
GUUGCUGUGGAUGUGACAGCAACGU



 



330
HPV6/11-na-7
CGGACGAUUCAGGUACAGAAAAUGA



 



331
HPV6/11-na-8
CAUUAUGCGACUGUGCAGGACCUAA



 



332
HPV6/11-na-1
ACAGCCAAAAAAGGUAAAGCGACGG



 



333
HPV6/11-na-2
GAAAAUGGGGGAGAUGGUCAGGAAA



 



334
HPV6/11-na-3
GAGGACGAGGAAGAUGGAAGCAAUA



 



335
HPV6/11-na-4
GGCAGCACAGUUAUAUGUUCUCCUG



 



336
HPV6/11-na-5
CUACUACAUACACCCCCGCACAGAC



 



337
HPV6/11-na-6
CUAUGGGAACACCCUUUAGUCCUGU



 



338
HPV6/11-L1-7
ACGCCGUAAACGUAUUCCCUUAUUU



 



339
HPV6/11-L1-1
UAGCGACAGCACAGUAUAUGUGCCU



 



340
HPV6/11-L1-2
CAGGCUUUGGUGCUAUGAAUUUUGC



 



341
HPV6/11-L1-3
CUGUGGUAGAUACCACACGCAGUAC



 



342
HPV6/11-L1-4
GAGUAACCUAAGGUCACACACCUGC



 



343
HPV6/11-L1-5
CCACACCCUACAUAUUUCCUUCUUA










B. Design of the Immobilization and Detection Probe
To design the immobilization and detection probes, all available HPV genomic sequences were aligned. From these alignments, the most closely related subgroups of HPV types according to phylogenetic tree classification were selected. These closely related HPV subgroups were re-aligned in smaller groups for full length, E6/E7 and L1 regions. Specific oligonucleotide probes for each HPV type were extracted from the nonconsensus regions of the realigned sequences. Probes were selected having 25 to 32 bp and a Tm from 55° C. to 70° C. The probes were then compared against other HPV types present in the NCBI Database using a BLAST search to confirm their uniqueness for each specific HPV type. Multiple probes were designed for each HPV type. A complete list of the probes generated according to this method is found in Table 2 (below). To protect against deletion or mutation causing a false positive in the assay, one probe each for the E6/E7 and L1 regions of the HPV genome was developed. This is especially helpful for integrated targets, as some regions can become disrupted during integration.
The immobilization probes are modified so as to facilitate binding to the detection beads. In the following examples, Luminex® microspheres are utilized as the detection bead, which are coated with carboxy groups to facilitate immobilization of capture probes. Therefore, the immobilization probes all contain a 5′Amino-C12 modification
In the following examples, SA-PE is used to detect captured nucleic acids. Accordingly, all of the detection probes in the following examples have a 5′ Biotin modification to facilitate detection by SA-PE.







TABLE 2







E6/E7 SPECIFIC IMMOBILIZATION/


DETECTION PROBES.












SEQ



HPV

ID



TYPE
PROBE ID
NO
PROBE SEQUENCE





HPV 2
E6_-2A
344
TGTATGGTGCAAACGGCCGTTATCAGAG


 



E6_-2B
345
ACATTGCATGAACTGCGGGTCATC


 


HPV 3
E6_-3A
346
TCTACTGTGCAGAAACACCGGAATAGGA


 



E6_-3B
347
TACGAAACAGCTGACTACAACTGAACTAC





AA


 



E6_-3C
348
TCTGGTCATTGGAGGGGGAGCTGTCAGTA





C


 



E6_-3D
349
CAGCTGACTACAACTGAACTACAAGC


 


HPV 6
E6_-6A
350
GGCTATCCATATGCAGCCTGCGCGTGC


 



E6_-6B
351
CAAGACATCTTAGACGTGCTAATTCGG


 



E6_-6C
352
CAAGACATTTTAGACGTGCTAATTCGG


 



E6_-6D
353
GGTAAAACATATACTAACCAAGGCGCGG


 



E6_-6E
354
GGTAAAACATATACTAACCAAGGCACGG


 


HPV 7
E6_-7A
355
ACAGCTAGAACTTTATTTGAATTATGTG


 



E6_-7B
356
TAACAGCATTTTACAAACAGCTGAGGTGC





TG


 



E6_-7C
357
AGCGTGTGTAAAGTGTTTAGAATTTTAT


 


HPV 10
E6_-10A
358
TGCAGAAGCTATGTCCATGGGTGCACAGG





A


 



E6_-10B
359
GCTTTTGTGTAGAAATTGTGGAATACCTT





TG


 



E6_-10C
360
GGCAGCATTTGCACTTAGAGAATTAT


 


HPV 11
E6_-11A
361
GTGTGCCTGTTGCTTAGAACTGCAAGGG


 



E6_-11B
362
ACTAAAGCACATATTGGGAAAGGCACGC


 



E6_-11C
363
GAGTGCACAGACGGAGACATCAGACAACT





AC


 


HPV 16
E6_-16A
364
CAGACATTTTATGCACCAAAAAAGAACT


 



E6_-16B
365
AGTTTGTATGGAACAACATTAGAACAGCA





AT


 



E6_-16C
366
CATAAAGTTACCAGATTTATGCACAGAGC


 



E6_-16D
367
CGATGTATGTCTTGTTGCAGATCATCA


 


HPV 18
E6_-18A
368
GCTACCTGATCTGTGCACGGAACTGAACA


 



E6_-18B
369
GCAAGACAGTATTGGAACTTACAG


 



E6_-18C
370
CCGAGCACGACAGGAACGACTCCAACGAC





GC


 


HPV 26
E6_-26A
371
AGAGAACGACCCAGAACGCTACATGAGC


 



E6_-26B
372
TGCAATTTGTGACCTAAGAGTAGTATATA





GAG


 



E6_-26C
373
ACGTTCGAGTGCTGGAGCAGATGTTAATG





GAA


 



E6_-26D
374
TCCTTGGTGTGCCATCAGTGTGCTGCACA





GT


 


HPV 27b
E6_-27A
375
ACACTGCATGCAGTGCGGGTCAAC


 



E6_-27B
376
GCGTGTATTGCAGACGAGCGCTTTCAGAC


 



E6_-27C
377
GCGTGTATTGCAGACGAGCGCTTTCAGAC


 



E6_-27D
378
AGCGCTTTCAGACGCTGATGTATT


 



E6_-27E
379
AGCGCTTTCAGACGCTGATGTATT


 


HPV 28
E6_-28A
380
GCACTGCATATTCTGCGCCAAAGTGC


 



E6_-28B
381
GCCAAAGTGCTGACCACAGCGGAGCTAT


 



E6_-28C
382
ACTGCAGGGCATTGTGCGACGCCTGAAGC





AC


 



E6_-28D
383
TGCATAGCTGGCTACTGGAGAGGGAGCTG





TC


 


HPV 29
E6_-29A
384
CAGCCCAGAACTGGCAGCATTTTGC


 



E6_-29B
385
CGCTGCTTATTGTTTGAAGGCATAAAGC


 



E6_-29C
386
GTGCCACAAGCCACTTGTCAGAGAGG


 



E6_-29D
387
CAAAATTTCTGGATACTGGAGAGGGAGTT





GC


 


HPV 30
E6_-30A
388
GCACCATCTTTGTGAGGTACAAGAAACAT





CG


 



E6_-30B
389
CAAGAAGGAATTATCCAGCTCAGAGG


 



E6_-30C
390
GACTGGTATATAGGGAGGACAGCCCA


 



E6_-30D
391
CACAACGTCCACTGAGACAGCAGTATAAT


 



E6_30-E
392
CTGCGTGCCCTACAACAGATGCTTATGGG





C


 


HPV 31
E6_31A
393
CTACTGCAAAGGTCAGTTAACAGAAA


 



E6_31B
394
CTACTGCAAAGGTCAGTTAACAGAAACA


 



E6_31C
395
TTGACAAACAAAGGTATATGTGATTTG


 



E6_31D
396
AAAAAGAAACGATTCCACAACATAGG


 


HPV 32
E6_-32A
397
ACCACTTAACCAGTGCTGAAGCGTATGCA


 



E6_-32B
398
GCATACAGTAGAACAAGAAACAGGACTAC





TG


 



E6_-32C
399
CCTGCCAACGTGTGACCCGACAACGTGC


 



E6_-32D
400
GCCAGTGTAGTAACCGGGGAAACACC


 


HPV 33
E6_-33A
401
CTGTGTTTGCGGTTCTTATCTAAAATTAG





TG


 



E6_-33B
402
CACAACATTGAACTACAGTGCGTGGAATG





C


 



E6_-33C
403
ATTATTCTGTATATGGACATACATTAGAA





CA


 



E6_-33D
404
ATTATTCTGTATATGGAAATACATTAGAA





CA


 



E6_-33E
405
TGTAAAAACGCCATGAGAGGACACAAGCC


 



E6_-33F
406
ACACAACATTGAACTACAGTGCGTGGA


 



E6_-33G
407
ACACCACATTGAACTACAGTGCGTGGA


 



E6_-33H
408
ATTATTCGCTATATGGAGAAACATTAGAA





CA


 



E6_-33I
409
CAGGATATAAATCTAAAACATATTC


 



E6_-33J
410
CAGGATGTAAATCTAAAATATATTC


 



E6_-33K
411
ATCTGCAAATGCAAAATCATATACCTCAG


 



E6_-33L
412
ATCTGCAAATACAAAGTCATATACCTCAG


 


HPV 34
E6_-34/64A
413
CAGCCTTATGTGAAGAGGTCAACATTTCA


 



E6_-34/64B
414
GCAGGACATTGTGTTAGATCTGAAACCAA





CG


 



E6_-34/64C
415
CACACGCTGACCTATTAGTGTTAGAAGAC





C


 


HPV 35
E6_-35A
416
AGAAGGCCAGCCATATGGAGTATGCATG


 



E6_-35B
417
GAAGAAAAAAAACGATTCCATAACATCGG


 



E6_-35C
418
ACAGAGCACACACATTGACATACGTAAAT





TGG


 


HPV 39
E6_-39A
419
TGCAGACGACCACTACAGCAAACCGAGG


 



E6_-39B
420
GCAGACGACCACTACAGCAAACCGAGG


 



E6_-39C
421
CCAGCAGAAAAATTAAGACACCTAAATAG





C


 



E6_-39D
422
AAGAGAAACCCAAGTATAACATCAGATAT





GCG


 



E6_-39E
423
CTAACACGAAGAGAAACCCAAGTATAACA





TC


 


HPV 40
E6_-40A
424
CAGGCCAGGACCCTGTATGAACTGTGTG


 



E6_-40B
425
AAGACGGTCCTAAAAACAGCTGAGGTACT





G


 



E6_-40C
426
CGCATGTCCACGGTGCCTGGACCTGCAC


 


HPV 42
E6_-42A
427
GCACTTAACAGGCGCAGAGGTGCTCGCG


 



E6_-42B
428
GTATACAGTGGAGAAAGAAACTGGACTAC





TT


 



E6_-42C
429
GTACAGCAGACACAGGTAGAACACGGAC


 


HPV 43
E6_-43A
430
CTTTGACTACGCAGCATATGCAGATACTG





T


 



E6_-43B
431
CAGTGTTTGATTTGTGCATTAGATGC


 



E6_-43C
432
ATCACCAGTGGAAAAAGTACAGCATA


 



E6_-43D
433
GCACATCCTGTCTGTGTGTAATTCGAC


 


HPV 44
E6_-44A
434
GAAAAACGTTAAGTACTGCAGAGGTTT


 



E6_-44B
435
TAAGTCAATTCTGGACGTGCTGATACG


 



E6_-44C
436
CCACCTGTGGTACATGTAGTCGGAAGG


 


HPV 45
E6_-45A
437
GCATTACAGGATGGCGCGCTT


 



E6_-45B
438
CCATTGAACCCAGCAGAAAAACG


 



E6_-45C
439
GTAGAGAGCTCGGCAGAGGACCTTAGAAC





AC


 


HPV 51
E6_-51A
440
GAAGCTTTGAACGTTTCTATGCACAATAT





A


 



E6_-51C
441
CAAAAATTAGAGAGTATAGACGTTATAGC





AGG


 



E6_-51D
442
ATGCGCTAATTGCTGGCAACGTACACGAC


 


HPV 52
E6_-52A
443
GAGGATCCAGCAACACGACCCCGGACCC


 



E6_-52B
444
GGCTGCAGTGTGTGCAGTGCAAAAAAGAG





C


 



E6_-52C
445
CCATATGGCGTGTGTATTATGTGCCTACG





C


 



E6_-52D
446
GATGAGGAGGATACAGATGGTGTGGACCG


 



E6_-52E
447
GAGGATCCAGCGACACGACCCCGG


 



E6_-52F
448
AGGCTGCAGTGTGTGCAGTGCAAAAAAGA





GC


 



E6_-52G
449
AGGCTGCAGTGTGTGCAGTGTAAAAAAGA





GC


 



E6_-52H
450
TGTGCAGTGCAAAAAAGAGCTACAACGAA





GA


 



E6_-52I
451
GGAAAACATTAGAAGAGAGGGTAAAAAAA





CCA


 



E6_-52J
452
GGAAAACATTAGAAGAGAGGGTAAGAAAA





CCA


 



E6_-52K
453
GGAAAACATTAGAAGAGAGGGTAAAAAGA





CCA


 



E6_-52L
454
GGAAAACATTAGAAGAGAGGTTAAAAAAA





CCA


 



E6_-52M
455
GGAAAACATTAGAAGAGAGGGTCGAAAAA





CCA


 


HPV 53
E6_-53A
456
TATATAATTTTGCATATACAGATCTAAGA





G


 



E6_-53B
457
GCAAGAAGGCATTGACAGCGTCAGAGG


 



E6_-53C
458
GTATAGAGACGGGTATCCGTATGG


 



E6_-53D
459
ATGGTATAGAGACGGGTATCCGTATGG


 


HPV 54
E6_-54A
460
GGGGGCAATGTCTGCTACTGAACCCCAC


 



E6_-54B
461
GCCTTTTGCAAGAAGACGGTGTGTACA


 



E6_-54C
462
GCTTGTGCACTGTGCCTAGAACTGCACGG





GC


 



E6_-54D
463
ACGGCTATGTGTGTATAGCACGCACACAG





G


 



E6_-54E
464
GGGGGCAATGTCTGCTACTGAAC


 



E6_-54F
465
GACGGTGTGTACAGCAGATATTTATGCA


 


HPV 55
E6_-55A
466
TAAATTACAGAATACCTGGAAGGGTCG


 



E6_-55B
467
CCACCTGTGGTACATGTAACCGGAACG


 


HPV 56
E6_-56A
468
TTGCAAAAAAGAACTAACACGTGCTGAGG


 



E6_-56B
469
AGTGTATAGGGATGATTTTCCTTATGC


 



E6_-56C
470
AACATCTAGAGAACCTAGAGAATCTA


 



E6_-56D
471
GAACTAACACGTGCTGAGGTATATAAT


 


HPV 58
E6_-58A
472
GCAATAAACACCATCTGCAATGGATGACC


 



E6_-58B
473
CCTGTAACAACGCCATGAGAGGAAACAAC





CCAACGC


 


HPV 59
E6_-59A
474
GTATGCAGCGTGTCTGAAATGCATTTCA


 



E6_-59B
475
GAACATTAGAGGCTGAAACCAAGACACC


 



E6_-59C
476
CATGAGCTGCTGATACGCTGTTATAGA


 



E6_-59D
477
CTTGTGTGCTACGAGCAATTACCTGACTC





CGA


 



E6_-59E
478
AACATTAGAGGCTGAAACCAAGACACC


 


HPV 61
E6_-61A
479
CCGTAGGGTCAGCAAAGCACACTCATCTA





T


 



E6_-61B
480
GCAGCAAACCGTTAAGTATACAGG


 



E6_-61C
481
AGCAAACCGTTAAGTATACAGGAAAAGGA





GC


 



E6_-61D
482
GCTACATGAACTACTGCTGGGCGACTTGT





CC


 


HPV 62
E6_-62A
483
TGTGGACCTGGACGACCTGCACCTA


 



E6_-62B
484
ACGGCGGTGGCAGCACTCATGCTTT


 



E6_-62C
485
GGAAAAGGAGTATCAGGTAGAGAGGGG


 


HPV 66
E6_-66A
486
GATTCCATATTCAGCAATACACAGGAA


 



E6_-66B
487
GATCCCATATTCAGCAATACACAGGAA


 



E6_-66C
488
CAAAAAGGAACTTACAAGTTTAGAGC


 



E6_-66D
489
AGTATATAGAAACAATTGGCCATATGC


 



E6_-66E
490
CCGGAGTATGGGGCAACATTAGAAAGTA


 



E6_-66F
491
GTATGGGGCAACATTAGAAAGTA


 



E6_-66G
492
TATATAGAAACAATTGGCCATATGC


 



E6_-66H
493
ATTAGTATATAGAAACAATTGGCCATATG





CAG


 


HPV 67
E6_-67A
494
CGGTAAATATATAAAGCACACCAGTGTCC





A


 



E6_-67B
495
CAGTGCAAGAAATATGTTTCAGGACACAG





A


 



E6_-67C
496
AAGTTTGCCCTGCGTGCAGTGCAAAAAAA


 



E6_-67D
497
CATTCACAGTACAGCAGCAGACGTCCGAA





C


 


HPV 68
E6_-68A
498
GCAGAAGGCAACTACAACGGACAGAGG


 



E6_-68B
499
TCAAGAAACACAAGTTTAAGTAACTATGC





A


 


HPV 69
E6_-69A
500
CGTCCGAGCGGTGGAGCAGCTGCTGATGG





GC


 



E6_-69B
501
GAGTTTGGTGTGCCACCAGTGTGCTACAT





AC


 



E6_-69C
502
GAGTTTGGTGTGCCACCAGTGTG


 



E6_-69D
503
ACGTCCGAGCGGTGGAGCAGCTGCTG


 


HPV 70
E6_-70A
504
CCCATACGGAATGGCGCGATTTCCCAAT


 



E6_-70B
505
ATAGTATATAGAAACGGGGAGCCATATGC


 



E6_-70C
506
ATAAATATAAATATGCATGGACCACGGCC





G


 



E6_-70D
507
CTCACAAGAGAACCTGCGATCTCTACT


 



E6_-70E
508
ACAAGTATAAATATAAATATGCATGGACC





ACG


 


HPV 71
E6_-71A
509
GTTTGCTGCATGTGCCTGCTGTTTGGAAA





T


 



E6_-71B
510
TAGACACCGGAACGCCAGTTACAGAGCAA





C


 



E6_-71C
511
AGAAAGAATAATTACAGAAGGCAGGCG


 


HPV 72
E6_-72A
512
ACGATACTGGACGTATTCGGGCTACGG


 



E6_-72B
513
GTCAGGAAAAGGAATATCAGGTGCAGACA





GG


 



E6_-72C
514
ATGAGAGGGACGGTGTTGGTGTGCAG


 


HPV 73
E6_-73A
515
AACCTGGACTGTGTGTTTTGCCAACGTGG


 



E6_-73B
516
GTATAGGCGATATAGACAATCAGTATATG





GCA


 



E6_-73C
517
ACTTTAGACCTGAAACCAACAACCGAAAT


 



E6_-73D
518
ACAAAGCTGATTTAAGAGTGATAGAAGAG





T


 


HPV 74
E6_-74A
519
CCATTTGCAGCGTGCGCCATTTGCTTA


 



E6_-74B
520
AAACTAGGCGACACCTGGAAAGGGCGCTGC


 



E6_-74C
521
GTGCAGTGTACAGGACCAGACATCAACAA





T


 


HPV 81
E6_-81A
522
GCTGGGGCCAGCAAATCCTACCAATTTGT





TT


 



E6_-81B
523
CGCAGCGTGCTTGTGCAGAGAAGCTAAAG





TAC


 



E6_-81C
524
GCGGCGGTGGCAATATTCGTGCTTCGGAC





CA


 


HPV 82
E6_-82A
525
CCACAAGTAAAGGACATAGTGTTGGAG


 



E6_-82B
526
GGTGGTGGACGACAAAAAAAGGTTTCAT


 



E6_-82C
527
GCCTGGTGGGCCCGTGTTGCGCGAACAAC





T


 


HPV 83
E6_-83A
528
CCACTGGCACAGCTGTATATACGATGCCA





T


 


HPV 84
E6_-84A
529
TGTGCTGTGCCAGGAATACGAGGTGGAGT





TCGACG


 



E6_-84B
530
AGGAAGAATTAACGGAAGGCGAAGTGC


 



E6_-84C
531
GTAAAGGAATTACTAATTGTTTGGAGG


 


HPV 85
E6_-85A
532
CCTATGCAACACACTGGACACATCACTGC


 



E6_-85B
533
GTTAGAAAAACTAACAAATAGCAATATAT


 



E6_-85C
534
CTGTATTGCTATGAGGAATTAAACAACTC





AG


 



E6_-85D
535
GACCTATGCAACACACTGGACACATCACT





GC


 


HPV 86
E6_-86A
536
CTAAAGGAATTATTACTGGTCTGGAAA


 



E6_-86B
537
CAAGACACAGGCGTATCATTGGCACACTT


 



E6_-86C
538
CTGCATATGGTGGAATTAAATCTGCAT


 


HPV 87
E6_-87A
539
TTAAGGGAATTATTGCTGGTGTGGAGA


 



E6_-87B
540
GGGAATTATTGCTGGTGTGGAGATTTGG


 



E6_-87C
541
GAGCATATGATACACGCGAATCTGCAC


 


HPV 89
E6_-89A
542
ATATTGCACCAAGGAGCTTACAAC


 



E6_-89B
543
GGCAGCTGCCCCATGGTGTATGTGCACCG


 



E6_-89C
544
CGGCCGCACGCCGACCATCCAGGAT


 



E6_-89D
545
CGTGTGGTGTGTGCTATCGTGCAGTTAGG


 


HPV 91
E6_-91A
546
GTACGCGGCATTAGCAGTAACAGTAGAG


 



E6_-91B
547
CGAGTGCACCTCTTGTTATTGTTCAATTC





GT


 



E6_-91C
548
GCACCTCTTGTTATTGTTCAATTCGTC


 


HPV 94
E6_-94A
549
GTGCTGCGTGTTCTGCACCAAACAGC


 



E6_-94B
550
CGTGTTCTGCACCAAACAGCTGACCGTAG





CC


 



E6_-94C
551
CAGCTGACCGTAGCCGAATTGACTGC


 



E6_-94D
552
CTGGAGAGGGTGTTGTGCTTATTGCTGGA





CAC
















TABLE 3







L1 SPECIFIC IMMOBILIZATION/DETECTION PROBES.












SEQ



HPV

ID



TYPE
PROBE ID
NO
PROBE SEQUENCE





HPV 2
L1_-2A
553
CGATGCTGATTTGTATGATCCAGATA





CCCA


 



L1_-2B
554
TCAGTTCCAACTCCAGGCAGTCATGTT


 



L1_-2C
555
CAAGCGCGCCGCTGTTTCGGGGACC





ACGC


 



L1_-2D
556
TCCCTGACCTTTTGGGATGTGGATC





TCAGT


 


HPV 3
L1_-3A
557
CCCCAAATCTTCTAATTCCAAGATG





GATATT


 



L1_-3B
558
AGCAGAATGCGTCACCGGGTGATTGT


 



L1_-3C
559
TCTAGAGCTTATTACTGCACCTATAC





AAG


 



L1_-3D
560
GTTGTACATTAAAGGTGACAGTCAGA





GCGGC


 


HPV 6
L1_-6A
561
AACAGTGTACTAATACACCTGTACAG





GC


 



L1_-6B
562
TCCTATTGACATATGTGGCACTACAT





GT


 



L1_-6C
563
TATAATTAAGGGTAGTGGAAATCGCA





CGT


 



L1_-6D
564
GCTGCCCCTAAACGTAAGCGCGCC


 


HPV 10
L1_-10A
565
GGAACCCACCTGCACAGGGCGATTGC





CC


 



L1_-10B
566
CAACGGTGGGGGGCGAGACGTTGGTA


 



L1_-10C
567
TACCAATATGTGCTTGTGTGTTCCTT





CT


 



L1_-10D
568
GCCTCCCCTGCCACTACGTATGACGC





C


 


HPV 11
L1_-11A
569
TTCATCCCTGTTTGACCCCACTACAC





AG


 



L1_-11B
570
AGTGGTGGGTATGGTGGTAATCCTGG





TCAG


 



L1_-11C
571
GGGTACACAATGTTCAAATACCTCTG





TACAAAA


 



L1_-11D
572
TGTTCCCCTTGATATTTGTGGAACTG





TCTGC


 


HPV 16
L1_-16A
573
AACATCCAGACTACTTGCAGTTGGA


 



L1_-16B
574
ATTTTACAATCCAGATACACAGCGGC





TG


 



L1_-16C
575
AGCAAATGCAGGTGTGGATAATAGA


 



L1_-16D
576
TCCCCATGTAACAATGTTGCAGTAAA





TCCA


 


HPV 18
L1_-18A
577
GCAGGTGGTGGCAATAAGCAGGATA


 



L1_-18B
578
GGCCTGTGCTGGAGTGGAAATTGGC


 



L1_-18C
579
CCATGCCGCCACGTCTAATGTTTCTG


 



L1_-18D
580
GTCTCCTGTACCTGGGCAATATGATG


 


HPV 26
L1_-26A
581
CCTGCAATAGTTGTGCATGGGGATA


 



L1_-26B
582
TGGCCAAAAGGCCGAAATTCCTAAG


 



L1_-26C
583
GACACTGACAACAGGGACAATGTTTCA


 



L1_-26D
584
GGAGCCCCCTACATCTTCTATTTAT


 



L1_-26E
585
AAACCTGCAATAGTTGTGCATGGGGA





TA


 



L1_-26F
586
GGCGGGGGCTGTTGGGGATGCTATA


 



L1_-26G
587
GGGGGCTGTTGGGGATGCTATA


 



L1_-26H
588
ACTGGCCAAAAGGCCGAAATTCCTAAG


 



L1_-26I
589
ATTAAAGGTGCTGAATCAGGCAGGGAG





CCC


 



L1_-26J
590
TAAGGCGGGGGCTGTTGGGGATGCTAT





ACCCACCAC


 



L1_-26K
591
CACTAACTTACCTGCAATAGTTGTGCAT





GGGGATA


 


HPV 27
L1_-27A
592
AAAACGCACCGCTGTTGCGGGGGCGG





CGG


 



L1_-27B
593
AGCTGAGGTGTCTGATAATACTAATT





ATAAA


 



L1_-27C
594
ACTATCTCGGACCCCGGCAGTCATGTG


 



L1_-27D
595
GGTAGCAATAATAGGTTGGCAGTGCC





TAAGGTG


 


HPV 28
L1_-28A
596
ATCATCCACTAACAAAGCAGATGTGCCC





AAA


 



L1_-28B
597
GTCAAAATACACAACAGGGAGATTGCCC





TCCG


 



L1_-28C
598
TATTACAGGCCAATAAATCGGACGTGCC





CT


 



L1_-28D
599
CAGGGCAACGGGAGGGATGTGATTGGT


 


HPV 29
L1_-29A
600
ACATTATTCAATTCCCAAATCCTCTGG





TA


 



L1_-29B
601
GGAGGTAGGTCGAGGGCAACCTCTCGG





TGTC


 



L1_-29C
602
CACTGTGTGTGCACGCACTAGTTCCGCT





GC


 



L1_-29D
603
GTTGTGTGCTACCACAGAGTCTCAACC





GTTG


 


HPV 30
L1_-30A
604
GCCCCTCAGGCCCCATTTGACACTACA


 



L1_-30B
605
GGCTGGTAATTCCAAAACAGATGTT


 



L1_-30C
606
AAATAACAGGGATCCCCCGCCAAGCT





CA


 



L1_-30D
607
TTCCTTACTATTTATTGTGCATGAAT





GTATG


 


HPV 31
L1_-31A
608
CAGTGCTAGGCTGCTTACAGTAGGC


 



L1_-31B
609
GACAATCCTAAAAAAATAGTTGTACCA





AAGGTG


 



L1_-31C
610
CCGGTGGTCCTGGCACTGATAATAGG


 



L1_-31D
611
TAGTCCTTGTAGTAACAATGCTATTA





CCCCT


 


HPV 32
L1_-32A
612
GCCATTAGATATTATGAACTCCATTAG


 



L1_-32B
613
GGACATGTATATAAAAGCTTCTAATGG


 



L1_-32C
614
TATCCAACTCCCAGTGGTTCTATGGT





CA


 



L1_-32D
615
CTGAAGACACATACAAGTCTACTAAC


 


HPV 33
L1_-33A
616
GCTAAAAAATTATTGGTACCCAAAGTA





TCA


 



L1_-33B
617
AGTATCCTGGACAACCGGGTGCTGAT





AAT


 



L1_-33C
618
CTTGGATGTAAGCCTCCAACAGGGGAA


 



L1_-33D
619
CACATCCACCCGCACATCGTCTGCA


 


HPV 34/64
L1_-34/64A
620
ACTAATGGGAAACGTAAGATTGCTGTA


 



L1_-34/64B
621
GTGGAAACATAGCAGATAGTAGGGAG


 



L1_-34/64C
622
AGGTACTGTAGGCGATGCTATTCCAGA





TGACT


 



L1_-34/64D
623
GTCTGCACCTTCATCATCTAGTACAG


 



L1_-34/64E
624
AAAGTGGAAACATAGCAGATAGTAGG





GAG


 


HPV 35
L1_-35A
625
CAGTTCTAGGCTATTAGCTGTGGGT





CAC


 



L1_-35B
626
GCAGTACCCAAGGTATCTGGTTTG


 



L1_-35C
627
ATCATTTTATGATCCCTGCCTCCA





GCGTT


 



L1_-35D
628
AAATATGTTGGTAACTCTGGTAACT





CTG


 


HPV 39
L1_-39A
629
ATATAGGGTATTTCGCGTGACATTG





CCC


 



L1_-39B
630
AAAGGCATGCAAGCCCAATAATGTAT





CTA


 



L1_-39C
631
ACGTGCAAACCCCGGTAGTTCTGTA





TACTG


 



L1_-39D
632
CAGTTTGGTAGACACTTACAGATACC


 


HPV 42
L1_-42A
633
CAAAAAGGCCAAATAAGACATCTATC





CCCAAA


 



L1_-42B
634
TAATTTATATAACCCAGATACGCAGC





GCA


 



L1_-42C
635
ACATATGGTGGAGGCCCTGGTACAG





AC


 



L1_-42D
636
ACTGTCTGTAGGTAAACGAAAGGCGT





CTAC


 


HPV 45
L1_-45A
637
GTACCTAATGGTGCAGGTAATAAACA





GGCTG


 



L1_-45B
638
GTTTAGAGTAGCTTTACCCGATCCT


 



L1_-45C
639
TTGGGCATGTGTAGGTATGGAAATT





GGT


 



L1_-45D
640
GCTCATGCAGCTACAGCTGTTATTA





CGC


 


HPV 51
L1_-51A
641
CCAAGCATTCTATTGTTATACTAGGT





GGGG


 



L1_-51B
642
CTCAACGCGTGCTGCTATTCCTAAA


 



L1_-51C
643
GTAATGGCCGTGACCCTATAGAAAG


 



L1_-51D
644
TATGTTAGTTTTTGTATGCTTGTGCA





CACT


 



L1_-51E
645
TTAACTATTAGCACTGCCACTGCTGC


 



L1_-51F
646
AACCTCAACGCGTGCTGCTATTCCT





AAA


 



L1_-51G
647
AACCTCAACGCGTGCTGCTATTCCT





AAAGTA


 


HPV 52
L1_-52A
648
TATTAAAAACACCAGTAGTGGTAA





TGGT


 



L1_-52B
649
AATATGCTGGTAAACCTGGTATAGAT





AAT


 



L1_-52C
650
AACCCCTTGTAATAATAATTCAGGAA


 



L1_-52D
651
CCTACAGCTCATTAACAGTGTAATAC


 


HPV 53
L1_-53A
652
ATAGCTATTCAGGATACTGCCCCGGAC


 



L1_-53B
653
CCCATTGGAACTTATCAATTCACCT





ATT


 



L1_-53C
654
CGTTATTGGTGAGGAAATACCTAAT





GAC


 



L1_-53D
655
CTTTCCGCAACCACACAGTCTATGTC


 


HPV 54
L1_-54A
656
TTAAAGTACAAAAAACCAATAATAA





GCAAAG


 



L1_-54B
657
CAACCTATGTACACCTAATACATTG





GCT


 



L1_-54C
658
AGTGAGGTACCCCTTGATGTAGCTA





CCTCA


 



L1_-54D
659
TACAGCATCCACGCAGGATAGCTTT





AATAA


 


HPV 56
L1_-56A
660
GACTAAGGACAATACCAAAACAA


 



L1_-56B
661
GTACTGCTACAGAACAGTTAAGTAA


 



L1_-56C
662
GCCAGTGGCCACCAGCCTAGAA


 



L1_-56D
663
ACTAGGTCAAAGCCTGCTGTAG


 



L1_-56E
664
AAAATCTGCTCCTACCTCCACCTCTA





CAC


 



L1_-56F
665
ATCTGCTCCTACCTCCACCTCTACAC


 


HPV 57
L1_-57A
666
GAGCTCTAGGCTCCTCACAGTAGG





CCAT


 



L1_-57B
667
GAAAAATAGCACTAATAAGGTGTCT





GTA


 



L1_-57C
668
CAACCTCTATGATCCCGACACCCAG





CGTCTG


 



L1_-57D
669
TGTCAAAAGTTCTACCGTCCAGACC





CCCGGT


 


HPV 58
L1_-58A
670
CAGTTCCAGACTTTTGGCTGTTGGCA


 



L1_-58B
671
CAGATATCCCGCACAGCCAGGGTCT


 



L1_-58C
672
CCCGGATGACCTTTATATTAAAGGG


 



L1_-58D
673
ATTACACTAACTGCAGAGATAATGAC


 


HPV 59
L1_-59A
674
AAAGGTGGTAATGGTAGACAGGATG


 



L1_-59B
675
AGCATCTGCTGTTGATACCAAAGATA





CACGT


 



L1_-59C
676
GACATACGTGCCAACCCAGGCAGTTA





TTTA


 



L1_-59D
677
CCCATCACCAAAACGTGTTAAGCGTC





GCAAG


 


HPV 66
L1_-66A
678
CCGTGAAATCAATCAATACCTTCGC


 



L1_-66B
679
CATTCCTACAGATTTGTATTGGAAG





GGTG


 



L1_-66C
680
TAGACCCCCTAGACCCAAGGCTAGT


 



L1_-66D
681
AAAGCACATTAACTAAATATGATGC





CCGTG


 


HPV 67
L1_-67A
682
TATTAGTGGACATCCATTACTTAA





TAAG


 



L1_-67B
683
ATAATAAATACCCTAGCCAGCCTG





GTA


 



L1_-67C
684
ACCTACAGATTTGTATTTTAAGGG





ATCT


 



L1_-67D
685
CACCTTCTTCTTCCTCTTCCTCCTCTG


 



L1_-67E
686
GGTAATTGTATGACTGTTGTGTGT


 


HPV 68
L1_-68A
687
AGTGTTCCTGAGTCTACATTATAT





AATCCA


 



L1_-68B
688
ATAAAAATCCTAAAGACAGTAGGGAC


 



L1_-68C
689
CTTGTAGATACATACCGCTACCTACAA


 



L1_-68D
690
GGACCAATTCCCATTAGGACGCAAA


 


HPV 69
L1_-69A
691
TCTGGTTCAACAGCAGAAATTCCTA





AAGTG


 



L1_-69B
692
CTCTCGATTATTAACTTTGGGTCA





TCCC


 



L1_-69C
693
CTGCTAATGCAGACACTGATAATAGG





GAC


 



L1_-69D
694
TAAAAATGCACAGTCTCAGGTACAG





CGTGGC


 


HPV 70
L1_-70A
695
GTTTGGCCTTCCGGATCCTTCCCTT


 



L1_-70B
696
GGATATACGTGAGCGTCCTGGTACTC


 



L1_-70C
697
TGCCTGCACCGAAACGGCCATACCTG


 



L1_-70D
698
GTCAGCTAAATCGTCTTCCTCAGCC


 


HPV 73
L1_-73A
699
CTGGACAAAATACAGATGGTAGAGAA


 



L1_-73B
700
ACTTCACAAACTGTTAATACTGGTG





AT


 



L1_-73C
701
TGGTGATACCGGTGATAAAATCCCA





GATGACC


 



L1_-73D
702
GGCTAGTAGCTCTACTACAACGTAT





GCC


 


HPV 82
L1_-82A
703
ACCAGTACACGTGCTGAAATACCT





AAG


 



L1_-82B
704
CCCTTTAGATATAGCTCAGTCCGTG





TGT


 



L1_-82C
705
GCATTACTATAATAGGGCCGGTGT





GGTT


 



L1_-82D
706
TACTGGTACTGGCCGTGACCCTATT





GG


 


HPV 84
L1_-84A
707
ACTAATGTGCAATATCGTGCGGGTG





ATTGC


 



L1_-84B
708
TTTGGATCTCTGCACCACTACCTGT


 



L1_-84C
709
TCAGTCTTTTTACCTTAAGGGG


 



L1_-84D
710
GGGCCGCCGCCGCCAAGCCTAAGGAC


 


HPV 85
L1_-85A
711
TACTTCTGTAGTTACACACGACACT





AGA


 



L1_-85B
712
CTGTAAGCCCGGTGCTGTGCAAACAG





GTGAC


 



L1_-85C
713
TGATAGGGCAACACCTGGAAGCTGT





ATT


 



L1_-85D
714
TGTGGTTGTTCCACAAAAAAAGGAT





CCA


 


HPV 86
L1_-86A
715
CCTGTTACTGTTTCCTCCAGCCCTG





GAC


 



L1_-86B
716
AAACCAGGGGACTGCCCCCCATTA


 



L1_-86C
717
CTCCACAAGTTTGGAGGATACCTACC





GT


 



L1_-86D
718
GGTGTTTTGGGAGGTTGACCTT


 


HPV 87
L1_-87A
719
CAAGACAGGGGATTGTCCACCATT





GCAA


 



L1_-87B
720
CGAAAAGTTACAGGAAAACAAGTCC


 



L1_-87C
721
CTATTTTTTGAAGGGGGCGTCGTCT


 



L1_-87D
722
TAACAAACCCTATTGGCTGCAGCGGG


 


HPV 94
L1_-94A
723
GGCCGGTGGTGACCAAAACGTTGG





TAG


 



L1_-94B
724
TGTGCGTCCCTTCTGATGCCTCCACC





GCC


 



L1_-94C
725
CCATCTCTGTCCGCAAACGCTCGGC





GACCG


 



L1_-94D
726
GGCCGGTGGTGACCAAAACGTTGGTA









C. Sample Purification via Hybrid Capture.
Cervical clinical swabs, liquid-based cytology samples, and urine all have been tested with the presently disclosed methods and determined to be compatible. In principle, any type of sample could be used.
A 50 μl aliquot of a sample is placed in a well of a polystyrene hybridization plate and mixed with 25 μl of alkali Denaturation Agent (DNR) available from Qiagen Gaithersburg, Inc. (Gaithersburg, Md.). The plate is sealed and shaken to mix, then incubated at 57.5° C. for 15 minutes shaking at 900 rpm to denature the nucleic acids in the sample.
Following denaturation, a purification probe mix comprising each of the purification probes prepared in Example 1 in low viscosity NextGen PD is added to each reaction to a final concentration of 1 nM. The plate was shaken to neutralize. A 0.02% solid paramagnetic bead stock in YT blocker is prepared, 25 μl of which is added to each reaction. The plate is then covered with clear sealer and incubated at 57.5° C. for 30 minutes with shaking at 900 rpm.
D. Amplification.
The plate resulting from Example 1(C) is placed on magnetic rack for 2 minutes. The supernatant is decanted and the plate was then blotted with clean low lint absorbent tissue, such as Kimwipes® (Kimberly-Clark Worldwide, Inc.). The beads are washed four times by adding 120 μl of whole genome amplification (WGA) Wash Buffer (available from Qiagen Gaithersburg, Inc. (Gaithersburg, Md.)) into each well, waiting 1-2 minutes, decanting, and blotting. The wash buffer is drawn off using a small volume multichannel. 20 μl of WGA Reaction Mix (set forth in Table 3 below) is then added to each well.







TABLE 3







WGA Reaction Mix with QIAGEN REPLI-g Midi RXN Mix










Reagents
Rxn (1X)






Tris-HCl, pH 7.5
 50 mM



MgC12
 15 mM



(NH4)2 SO4
 10 mM



KCl
 50 mM



dNTP
 4 mM total



Primer
250 μM



REPLI-g
 0.5 μl/20 μl





*0.50 μl REPLI-g Midi per 200 μl reaction


*dNTP and Primers should be vortexed well prior to addition to the reaction mixture.






The plate is shaken to mix, then incubated at 30° C. for 2 hours. The amplicon may be stored at −20° C. or detection may directly be undertaken.
E. Genotvping
A 5 μl aliquot of the amplicon generated in Example 1D is transferred to a new round-bottom 96 well plate and mixed with 10 μl of 0.75×DNR. The plate then is incubated at 70° C. for 30 minutes to denature any nucleic acids.
After denaturation, 5 μl of a 5 nM stock of each detection probe heated to 70° C. is added to each well and the plate is incubated for 2 minutes at 70° C. A 10 μl aliquot of 0.75× HC2-Probe diluent (available from Qiagen Gaithersburg, Inc. (Gaithersburg, Md.)) is added, then mixed on a plate shaker at 800 rpm for 1 minute at room temperature. A 5 μl aliquot of a Luminex® microsphere solution at 1000 beads/μl (5000 beads total/assay) is added to each well, then the plate incubated at 50° C. for 30 minutes with shaking at 400-450 rpm. A 10 μl aliquot of a streptavidin-phycoerythrin solution (prepared in 10% Goat Serum in 1× phosphate buffered saline containing 3% TWEEN-20) is added to each well to an SA-PE final concentration of 400 ng/well). The plate is then incubated at 50° C. for 10 minutes with shaking at 400-450 rpm. Finally, the reaction mixture is diluted by adding 150 μl H2O to each well.
Thus, each Luminex® microsphere is conjugated with two oligonucleotide probes (one each in the E6/E7 and L1 regions) which are specific to that bead's HPV type. Each bead specific for an HPV type bears a unique fluorescent label. Target detection is achieved by binding specific biotinylated probes to each captured target. SA-PE bound to the biotinylated probe and a fluorescent signal is observed after excitation of the phycoerythrin by a laser. The phycoerythrin label and the fluorescent label of each bead were then independently measured on a luminometer. Measurement of the bead label indicates the genotype of the target nucleic acid bound to each bead. Measurement of the phycoerythrin label is used to determine the relative amount of target bound to each bead. The fluorescent data is then compiled to indicate the relative amounts of each target nucleic acid present in the sample.
Example 2
Demonstration of the Effect of Hybrid Capture Sample Preparation on the Detection of HPV Nucleic Acids
The following example demonstrates the inhibitory effect of human genomic DNA on detection of HPV nucleic acids using the method described at Example 1.
An 0, 102, or 103 copies of a plasmid comprising the HPV 16 genome were mixed with 0, 50, 100, 200, 1000, or 5000 ng of human genomic DNA. One set of samples was subjected to hybrid capture purification, while the other was not. After hybrid capture, the samples were amplified by whole genome amplification and the presence or absence of HPV 16 was determined. Results without hybrid capture sample preparation are shown at FIG. 1A, while results with hybrid capture sample preparation are shown at FIG. 1B. As can be seen, the hybrid capture sample prep efficiently removed inhibiting DNA, enabling whole genome amplification to proceed efficiently. This demonstrates that, under conventional methods, conventional amplification of the HPV target is reduced when extraneous human genomic DNA is present, due to wasteful amplification of undesired target.
Example 3
Detection of Quadruple Infections
Samples comprising a quadruple infection were tested in a 20-plex assay as set forth Example 1, utilizing hybrid capture, immobilization, and detection probes for HPV types 6, 11, 16, 18, 31, 33, 45, 34, 35, 52, 53, 58, 59, 66, 67, 68, 69, 70, 73, and 82. The samples tested had one of the following quadruple infections: (1) HPV 6, 11, 16, 18; (2) HPV 31, 33, 45, 34; (3) HPV 35, 52, 53, 58; HPV 59, 66, 67, 68; and HPV 69, 70, 73, and 82. Results are illustrated in FIG. 2. Under the methods described above, detection of quadruple infections from 102 to 107 of each HPV type can be simultaneously detected in a 20-plex reaction with good specificity and sensitivity.
Example 4
Detection with Small Amplicon Volumes
HPV 16 was tested for according to Example 1, except that 1, 2, 5, 7, and 20 μl of amplicon were individually used. The results are illustrated in FIG. 3A. The results show that a small amount of amplicon required for robust detection of low copy numbers. Amplification is very robust, and only a small portion of amplicon is required to saturate the detection method being used. Small volumes of amplicon gives strong signal both at low-copy amplicons, as well as high-copy (>107) amplicon.
The process was repeated with 0.5 and 5 μl over night. The results are illustrated in FIG. 3B. As is evident, even smaller amounts of amplicon can be effectively used if amplification proceeds overnight.
Example 5
Specific Detection
The process outlined in Example 1 was repeated for a sample containing both HPV types 33 and 58. An identical detection probe was used (consensus for HPV 33 and HPV 58), and either HPV 33 or HPV 58 capture probe was used when in the presence of both amplicons. When using the HPV 33 capture probe, only HPV 33 is detected (see FIG. 4A). When using the HPV 58 detection probe, only HPV 58 is detected (see FIG. 4B). The results for each capture probe are illustrated in FIGS. 4A and 4B. Thus, detection of the specific amplicon only occurred on the correct capture bead, regardless of the fact that the detection probe was bound to both amplicons.
Example 6
Sensitivity of Multiplex Experiments
Multiplex experiments to test the sensitivity of 26 HPV types by repeating the processes outlined in Example 1 for HPV types 6, 11, 16, 18, 26, 31, 33, 34, 35, 39, 45, 51, 52, 53, 54, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, and 85. The results are demonstrated in FIG. 5. These results show assay sensitivity at 1000 copies for each of the tested types. Specifically that an S/N of greater than 2 was achieved for all 26 HPV types.
Example 7
Specificity of Multiplex Experiments
Multiplex experiments were performed to test the sensitivity of the disclosed processes for 26 HPV types by repeating the processes outlined in Example 1 for samples comprising HPV types 6, 11, 16, 18, 26, 31, 33, 34, 35, 39, 45, 51, 52, 53, 54, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, or 85. The results are demonstrated in FIG. 6. These results show that all HR-HPV types were specific against other types at up to 106 copies. As can be seen, the S/N of all non-specific HPV types was less than 2.0 for each data point tested. With only one exception, the S/N of all specific HPV types was approximately 5 or greater.
Example 8
Detection of a Single HPV Infection in a Clinical Sample
The processes outlined in Example 1 were conducted on a clinical sample which was by a reference test shown to have an HPV type 16 infection. The results are represented in FIG. 7 and show successful detection of HPV 16.
Example 9
Detection of a Single HPV Infection in a Clinical Sample
The processes outlined in Example 1 were conducted on a clinical sample which was by a reference test shown to have an HPV type 18 infection. The results are represented in FIG. 8 and show successful detection of HPV 18.
Example 10
Detection of a Quadruple HPV Infection in a Clinical Sample
The processes outlined in Example 1 were conducted on a clinical sample which was by reference tests shown to have HPV type 16, 51, 59, and 82 infections. The results are represented in FIG. 9 and show successful detection of HPV 16, 51, 59, and 82.
Example 11
Detection of a Double HPV Infection in a Clinical Sample
The process outlined in Example 1 was conducted on a clinical sample which was by reference tests shown to have an HPV types 18 and 70 infections. The results are represented in FIG. 10 and show successful detection of HPV 18 and 70.
It will be appreciated that various of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, and are also intended to be encompassed by the following claims.

## Claims

1. A nucleic acid probe set consisting of:
a) a first nucleic acid probe consisting of one of the nucleotide sequences selected from the group consisting of SEQ ID NO: 102 to SEQ ID NO: 113, and complements thereof and a detectable label, wherein said first nucleic acid probe is capable of hybridizing under selective stringency conditions to a portion of a gene selected from the group consisting of E6 and E7; and
b) a second nucleic acid probe consisting of one of the nucleotide sequences selected from the group consisting of SEQ ID NO: 577 to SEQ ID NO: 580, wherein said second nucleic acid probe is capable of hybridizing under selective stringency conditions to a portion of an L1 gene;
wherein the first nucleic acid probe and second nucleic acid probe are capable of hybridizing under stringent conditions to the same HPV genome, and
wherein the nucleic acid probe set is capable of isolating HPV nucleic acids from non-HPV nucleic acids in a sample.

2. The nucleic acid probe set of claim 1, wherein the first nucleic acid probe further comprises a ligand.

3. The nucleic acid probe set of claim 1, wherein said nucleic acid probe set further comprises:
(a) a purification probe set, wherein the probes of the purification probe set comprise at least one consensus probe capable of hybridizing to a plurality of target nucleic acids from different HPV types.

4. The nucleic acid probe set of claim 3, wherein the probe set further comprises:
(b) an immobilization probe set comprising at least one polynucleotide probe specific for one of the plurality of target nucleic acids.

5. The nucleic acid probe set of claim 4, wherein the immobilization probe set comprises at least one probe specific for each of the plurality of target nucleic acids.

6. The nucleic acid probe set of claim 4, wherein the probe set further comprises:
(c) a detection probe set comprising at least one polynucleotide probe specific for one of the plurality of target nucleic acids.

7. A kit for genotyping a Human Papillomavirus nucleic acid, said kit comprising:
(a) the nucleic acid probe set according to claim 1;
(b) a nucleic acid polymerase;
(c) a primer;
(d) a first solid support;
(e) an anti-DNA:RNA hybrid antibody bound to or adapted to be bound to the first solid support; and
(f) a detectably labeled second solid support.