# Patent

## Title
Materials and methods for detection of HPV nucleic acids

## Abstract

Provided are nucleic acids capable of hybridizing to HPV 16 and/or HPV 18 nucleic acids, in particular, mRNA encoding E2 and E6-7 gene products. Such nucleic acids are useful in methods of isolating RNA from a biological sample, methods and means for determining the presence of particular RNA splice-form variants in a biological sample, methods and means for determining the relative ratio of RNA ratios in a biological sample, methods and means for predicting the progression of precancerous cervical lesions, and methods and means for detecting disruption of genes or gene expression.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/446,306, filed on Feb. 24, 2011, and also U.S. Provisional Application No. 61/486,118, filed on May 13, 2011, which are both hereby incorporated by reference in their entirety.


BACKGROUND
1. Field
The present disclosure relates to methods, compositions, and kits for determining the presence of a nucleic acid in a sample, including nucleic acids derived from Human papillomavirus (“HPV”).
2. Description of Related Art
Human papillomavirus (HPV) infection is the most important cause of cervical cancer, 13 types of which cause HPV-related cervical disease and cancer. Screening for oncogenic HPV DNA using molecular tests has been useful to diagnose HPV-related disease. However, current testing methods cannot precisely predict which infections may develop into cancer because most HPV infections are transient and regress and clear spontaneously. Therefore, additional biomarkers are being explored for use in reflex assays to confirm which infections will progress and require further treatment.
The progression of disease may be related to the expression of certain HPV genes. Detection of HPV mRNA may, therefore, be an additional biomarker for severe infections. Some HPV mRNA assays being developed for diagnostics detect a single type of transcript species, such as the E6 or E7 oncogenic sequences. These assays may not predict severe infections because the abundance of a single species may fluctuate due to the complex pattern of expression that occurs during the course of disease, or due to degradation of HPV from immune responses. In addition, an mRNA target may degrade after collection, or the number of infected cells in the collected specimen may be low, both of which may affect the assay result. As a solution, HPV assays designed to detect simultaneously two species of mRNAs in a ratio may be more predictive of disease than assays that detect a single mRNA species.
Additionally, HPV DNA is typically maintained as a productive infection in a circular, episomal state at 50-100 copies per cell. In this state, transcription of the HPV oncogenes E6 and E7 is tightly controlled by the E2 protein. E6 and E7 target p53 and pRb, respectively, and thus interfere with the normal cell cycle. Cells in which this transcriptional control is removed have a proliferative advantage over other cells due to their accelerated reentry into the cell cycle. Disruption or deletion of the E2 gene, as frequently occurs during integration of the virus into the host genome, removes the negative feedback on E6 and E7, activates telomerase, and derepresses hTERT expression, and thus clearly contributes to the progression of cell immortalization and ultimately, cancer progression.
The detection and characterization of specific nucleic acid sequences and sequence changes have been utilized to detect the presence of viral or bacterial nucleic acid sequences indicative of an infection, the presence of variants or alleles of mammalian genes associated with disease and cancers, and the identification of the source of nucleic acids found in forensic samples, as well as in paternity determinations. Characterization of the RNA species involved in normal biological processes may be important to understanding various little known biological processes.
The detection and characterization of RNA (e.g., messenger RNA, transfer RNA, ribosomal RNA, small nuclear RNA, and other RNAs) is an important tool in many fields including molecular biology, toxicology, and biochemistry. Messenger RNA (mRNA) is an essential functional constituent of a cell; during the process of gene expression, the functional single strand structure of mRNA is synthesized and serves as an intermediate template for the translation process in protein synthesis. The brief existence of an mRNA molecule begins with transcription of DNA into an RNA molecule, and ultimately ends in degradation. During its life, an mRNA molecule may also be processed, edited, and transported prior to translation. Splicing is the process by which pre-mRNA is modified to remove certain stretches of non-coding sequences called introns; the stretches that remain may include protein-coding sequences and are called exons. Sometimes pre-mRNA messages may be spliced in several different ways, allowing a single transcript to encode multiple proteins.
Detection of messenger RNA (mRNA) is critical in diagnostics because it can provide viral load and gene expression information that DNA detection cannot. These factors often give clues about the progression and prognosis of a disease. The current technologies for mRNA detection present a number of problems including complexity and potential for contamination.
The most common methods of mRNA detection include Northern blot, ribonuclease protection assay (RPA), and reverse-transcriptase polymerase chain reaction (RT-PCR). However, each of these techniques, while affording some advantages in sensitivity, requires time and material demands. In addition, some techniques require amplification of the target mRNA since total mRNA represents only about 1% of the total RNA and any particular mRNA is a significantly smaller percentage.
Currently, reverse transcriptase-polymerase chain reaction (RT-PCR) is widely used to characterize RNA transcripts. However the method has the following limitations: 1) only a limited number of the specific regions can be co-amplified; 2) mutations or alternative splicing can limit the ability of specific primers to detect the RNA; and 3) it is difficult to characterize the mRNA structure in a continuous mode method.
It therefore would be useful to have materials and methods capable of determining whether the a given nucleic acid is present or absent in a sample. Additionally, it would be useful to have materials and methods capable of determining whether a gene—including the HPV E2 gene—is disrupted, deleted, or otherwise is not being expressed in a host cell.
BRIEF SUMMARY
The present disclosure provides nucleic acids and methods useful in detecting specific nucleic acids in a sample and determining whether those nucleic acids are intact or disrupted.
In an aspect, an isolated nucleic acid is provided, having an overall length of not more than 200 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: 105 and SEQ ID NO: 111 to SEQ ID NO: 308, RNA equivalents thereof, and a complements thereof.
In an aspect, a method of detecting the presence of a target RNA is provided, the method comprising: a) providing at least one DNA capture probe, wherein the at least one DNA capture probe is bound to a support; b) hybridizing the target RNA to said at least one DNA capture probe, yielding a target RNA:DNA capture probe complex; c) isolating the target RNA:DNA capture probe complex; d) providing at least one DNA amplification probe, and hybridizing said at least one DNA amplification probe to said target RNA:DNA capture probe complex, yielding a target RNA:DNA capture/amplification probe complex; e) providing an anti-RNA:DNA hybrid antibody, and incubating said target RNA:DNA capture/amplification probe complex with said antibody, yielding a target RNA:DNA:antibody complex; f) detecting said antibody, wherein said detecting indicates the presence of said target RNA. In one aspect, antibody is conjugated to a detectable marker, and the step of detecting comprises detecting the marker. In one aspect, the detectable marker is selected from the group consisting of alkaline phosphatase and horseradish peroxidase. In one aspect, the step of detecting comprises providing a second antibody that binds to said anti-RNA:DNA hybrid antibody, wherein said second antibody is conjugated to a detectable marker, and wherein said detecting further comprises detecting the marker. In one aspect, the support comprises a magnetic bead. In one aspect, the magnetic bead is conjugated to at least one streptavidin molecule, and the at least one DNA capture probe is conjugated to a biotin molecule. In one aspect, at least one of the capture probes and/or amplification probes is a nucleic acid probe as set forth above.
In one aspect, the at least one DNA capture probe and the at least one DNA amplification probe are from about 15 to about 200 bases in length.
In one aspect, the target RNA is a splice variant, and the at least one DNA capture probe and the at least one DNA amplification probe are selected to detect the presence of said splice variant.
In one aspect, the at least one DNA capture probe and the at least one DNA amplification probe are complementary to RNA from HPV high risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 66, 73, and 82.
In another aspect, a kit for the detection of a target RNA is provided, the kit comprising: a) at least one DNA capture probe, bound to a magnetic support; b) at least one DNA amplification probe; c) an anti-RNA:DNA hybrid antibody; and d) a detection reagent. In one aspect, said anti-RNA:DNA hybrid antibody is conjugated to a detectable marker, and said detection reagent comprises a substrate for said detectable marker. In one aspect, the kit further comprises a second antibody that binds to said anti-RNA:DNA hybrid antibody, wherein said second antibody is conjugated to a detectable marker, and wherein said detection reagent comprises a substrate for said detectable marker.
The present disclosure provides a method of providing target RNA for detection, the method comprising: incubating a biological sample containing the target RNA with carboxyl beads; isolating the beads; lysing the biological sample attached to the isolated beads; and isolating the beads from the lysed biological sample, wherein the resulting supernatant contains the target RNA for detection.
In another aspect, a method for nucleic acid detection is disclosed that does not rely on target amplification. Nucleic acids of interest are captured by specific nucleic oligonucleotides. Signal amplification is provided by adding DNA probes that cover the captured RNA target (or vice versa of the target is DNA) that is then detected using entities capable of binding specifically to DNA:RNA hybrids. This hybrid capture assay gives linear increases in signal as both quantity and length of transcripts increase. As a result, it can be used to measure deletions that existing technologies cannot. By assaying the extent of target nucleic acid disruption, as compared to total signal from a complete set of reference nucleic acids, one is able to whether, and the extent to which, the target is disrupted.
In an aspect, disruption of the target is determined by separating a sample into at least a first and second portion. The first portion of the sample is treated under conditions sufficient to generate two sets of DNA:RNA hybrids: one set comprising the target nucleic acid and one set comprising at least one reference nucleic acid. The second portion of the sample is then treated under conditions sufficient to generate the set of DNA:RNA hybrids comprising the reference nucleic acid, but not set comprising the target nucleic acid. The total amount of DNA:RNA hybrid in the first portion of the sample is then compared to the total amount of DNA:RNA hybrid in the second portion of the sample. If the target nucleic acid is missing, there should be the same amount of DNA:RNA hybrid in the first and second portions of the sample. Variations of the method also are presented for determining the extent of disruption, if any, by applying a plurality of probes specific for a substantial portion of the target nucleic acid and progressively removing the probes. The more probes that can be removed before a change in DNA:RNA hybrids is detected, the greater the extent to which the target nucleic acid is disrupted.
In another aspect, a method is provided to determine whether or not an E2 gene, cDNA, or mRNA is absent or disrupted. Such a method can be applied to, inter alia, determine whether the E2 gene is being expressed, whether the HPV genome is integrated into the host cell genome, assessing the progression of an HPV infection, and/or determining the risk of an HPV infection progressing to cancer.



BRIEF DESCRIPTION OF THE DRAWINGS
For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements.
FIG. 1 is a schematic diagram of target RNA (crosshatched bar) captured by biotinylated DNA probes (white bar). “B” represents a biotin moiety; “SA” represents a streptavidin moiety; “AP” represents alkaline phosphatase conjugated to an antibody, but AP could be any other appropriate detectable moiety (e.g., horseradish peroxidase, etc.), and B and SA could be replaced by other linkage moieties.
FIG. 2 is a diagram depicting the use of DNA capture probe (white bar), multiple DNA amplification probes (black bars), and multiple DNA:RNA hybrid antibodies to “amplify” the signal without the need for amplification of the target RNA (crosshatched bar). “B” represents a biotin moiety; “SA” represents a streptavidin moiety, B and SA may be replaced with other conjugation technology in which DNA probes are conjugated to the bead; “AP” represents alkaline phosphatase conjugated to an antibody, but AP could be any other appropriate detectable moiety (e.g., horseradish peroxidase, etc.).
FIG. 3 is a diagram of target RNAs (dashed arrows) captured by different DNA capture probes bound to a substrate (S). Non-conjugated DNA amplification probes (black bars) and multiple antibodies that detect and bind to DNA:RNA hybrid regions (conjugated to alkaline phosphatase or any other appropriate detectable moiety, such as horseradish peroxidase, etc.) are also shown. The substrate (e.g., a bead) may bear multiple DNA capture probes, and the DNA capture probes may be the same (i.e., the same sequence and/or length) or different (i.e., different sequences and/or different lengths).
FIG. 4 provides the results of an experiment showing the effect of adding unbiotinylated DNA probes after RNA capture. In this experiment, a variable number of biotinylated probes were conjugated to streptavidin beads. The target was the E6/7 gene transcript of HPV 16. The assay was performed with each set of beads with (black bars) and without (white bars) the addition of unlabeled signal amplification probes (one- versus two-step assay). When no signal amplification step was added (white bars), the signal increased with the amount of coverage provided by the capture probes. However, when signal amplification probes were added (black bars), the signal was greater than if they were not added, and they enable a higher signal with fewer (3-5) capture probes.
FIG. 5 shows that endogenous hybrids are often the source of clinical background noise. “RLU”=relative luminescence unit.
FIG. 6 shows the effect of lysis buffer (wherein 100% buffer contains about 3 M guanidine thiocyanate and about 2% detergent) concentration on assay background when assaying cellular samples in PreservCyt® Solution, and demonstrates that clinical background decreases with decreasing concentrations of lysis buffer.
FIG. 7 shows that hypotonic lysis of cell pellets ensures that background noise remains low and stable, and that the background does not change significantly regardless of the amount of specimen used. “PC”=PreservCyt® Solution; “PC(−)”=Specimen (cervical scrape) pool fixed in PreservCyt® Solution with no HPV target.
FIG. 8 shows limit of detection of HPV E6/E7 from HPV positive cells (SiHa). This shows that using the methods of the present disclosure, as little as 1×103 cells are required for HPV E6/7 RNA detection.
FIG. 9 shows results from tests of various lysis buffers for the ability to lyse cells captured by COOH beads. The data of FIG. 9, along with that of TABLE 1, below, shows the preferred lysis buffer is about 1M guanidine thiocyanate and about 0.7% detergent.
FIG. 10 shows cell capture by magnetic carboxylate-modified (COOH) beads (Sera Dyn catalog number 6515-2105-050350), over time, demonstrating that about 95% of the cells have been captured after incubation of 30 minutes.
FIG. 11 shows comparison of COOH bead capture with hypotonic lysis, and indicates that COOH bead capture is more efficient than hypotonic lysis for obtaining mRNA from cells. “PC-” indicates a pool of cervical scrape specimens that lack presence of HPV.
FIG. 12 is a diagram depicting capture and signal amplification probe design regions. The length of HPV transcripts can be “characterized” by capture onto magnetic beads with specific capture oligos that capture specific targets and detected with various sets of unlabeled oligonucleotides used to extend the length of the hybrid region. Signal will result if the capture RNA bears the sequence that is complementary to the capture probes that are used. Signal output will increase with successive addition of amplification signal probes until maximum length is reached where the signal will plateau. The various HPV transcripts for HPV 16 are shown. The regions denoted by the dashed boxes are designated for probe design.
FIG. 13 shows increasing signal as the number of signal amplification probes is increased. In this way, an RNA transcript length may be measured by the increasing signal generated by the increased number of consecutive amplification probes. In FIG. 13, each set of 5 oligos are adjacent to one another and result in the RNA:DNA hybrid getting longer, and signal stronger, as successive sets are added.
FIG. 14 shows that a fraction of cells with a high early:late HPV mRNA ratio may be detected against a background of cells with a low ratio. For this FIG. 14, SiHa cells (cervical cancer cell line) were added to a pool of cervical specimens (each diagnosed with a high-grade HPV-related lesion). The SiHa cells incorporate a high ratio of HPV early transcripts:HPV late transcripts, which is a common characteristic of cervical cancer. The sample mimicked a specimen that has cancer cells among pre-cancerous lesion cells. The results show that the invented assay will detect cancer cells in a pool of more benign lesion cells.
FIG. 15 shows HPV RNA stability of SiHa cells preserved in a LBC clinical specimen pool. The RT-PCR plots show the assay signal (y-axis) plotted against PCR cycle number (x-axis) for samples of SiHa cells incubated over the course of 67 days. Symbols are star, 3 days; square, 13 days; triangle, 26 days; filled diamond, 42 days; open diamond, 67 days. Values are an average of two reactions for each day.
FIG. 16a shows a general scheme for hybrid capture detection of HPV mRNA. HPV mRNA target (dotted line) is annealed to capture oligos (short grey bars) that are coupled to a magnetic bead (circle). The RNA target is annealed with signal amplification oligos (short black bars) to create a longer hybrid. The RNA:DNA hybrid is bound with a hybrid capture antibody conjugated with alkaline phosphatase (Y-shaped AP symbol). A chemiluminescent substrate (not shown) is added to detect the complex in a luminometer.
FIG. 16b shows a schematic of the HPV genome structure with labeled genes (large grey arrows). The loci for E6-7 probes (1) or E2 probes (2) are shown by black bars underneath. The arrangement of genes and the loci for DNA probes are similar for HPV 16 and HPV 18, but the primary sequences are unique.
FIG. 17a shows the dependence of luminescence signal output (average RLU, n=4) on the number of complementary signal amplification probes per assay for the same target input (1×105 copies, HPV 16 E6-7 in vitro transcribed RNA). In this experiment, the hybrid length increased in wells with the addition of 5, 10 and 15 probes. The signal did not increase for the well (labeled 5+15) with 5 non-complementary probes added to 15 complementary probes.
FIG. 17b shows the dependence of luminescence signal output (average RLU, n=3 samples, error bars show standard deviation) on target input (RNA copies per reaction) for a hybrid capture assay. Signal:noise ratio is given above bars.
FIG. 18a shows the dependence of signal:noise (average, n=3) on number of SiHa cells per assay was plotted for the HPV 16 E6-7 (grey bars) and E2 (black bars); for the two assays in separate wells. For these assays, the background noise was obtained from the signal from a control assay with no target added, approximately 50 RLU.
FIG. 18b shows the signal: noise values for HPV E6-7 and E2 mRNA assays were plotted as a ratio for the cancer cell lines, SiHa, Caski and HeLa; bars represent the average ratios of three replicate experiments.
FIG. 19 shows detection of the HPV 16 E6-7:E2 transcript ratio in a mixture of SiHa cells with the cells from a pool of HPV-positive specimens. Cultured SiHa cells were mixed with a pool (2 ml) of HPV-positive, liquid-based cytology specimens (approximately 100,000 total cells in 2 ml).
FIG. 20 shows the HPV 16 E6-7:E2 ratio in cervical specimens. The E6-7:E2 ratio was plotted from the hybrid capture assay results.
FIG. 21 illustrates a method for determining whether or not E2 gene expression is absent or disrupted.
FIG. 22 illustrates a comparison of the integrity of E2 gene expression in SiHa and W12 cells.
FIG. 23 illustrates a comparison of the integrity of E2 gene expression in LSIL and HSIL samples.



DETAILED DESCRIPTION
Before the subject disclosure is further described, it is to be understood that the disclosure is not limited to the particular aspects of the disclosure described below, as variations of the particular aspects may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular aspects, and is not intended to be limiting.
In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.
Isolated Nucleic Acids and Probes Capable of Hybridizing to HPV 16 and/or HPV 18
Nucleic acids consisting of not more than 200 nucleotides and being capable of hybridizing to HPV 16 or HPV 18 DNA or RNA are provided herein.
In an aspect, the nucleic acid comprises, consists essentially of, or consists of at least one nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 105 and SEQ ID NO: 111 to SEQ ID NO: 308, RNA equivalents thereof, and complements thereof. In a further aspect, the nucleic acid comprises, consists, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 105 and SEQ ID NO: 111 to SEQ ID NO: 308, RNA equivalents thereof, and complements thereof,
In an aspect, the nucleic acid is capable of hybridizing under stringent conditions to a nucleic acid 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 an HPV16 or HPV18 genome or a nucleic acid derived from the same. The sequence of an exemplary HPV 16 genome is disclosed at GenBank NC_01526 (SEQ ID NO: 106). The sequence of an exemplary HPV 18 genome is disclosed at GenBank X05015 (SEQ ID NO: 107).
In another aspect, the nucleic acid is capable of hybridizing or binding to a nucleic acid 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 99%, or 100% identical to an HPV16 or HPV18 mRNA or a complement thereof. In another aspect, the HPV 16 or HPV 18 mRNA is selected from the group consisting of E2 and E6/E7 mRNA.
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, a probe set is provided, said probe set comprising at least one of the isolated nucleic acids disclosed herein. By way of example and not limitation, the probe set may comprise an isolated nucleic acid comprising, consisting essentially of, or consisting of at least one nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 105 and SEQ ID NO: 111 to SEQ ID NO: 308, RNA equivalents thereof, and complements thereof. In a further aspect, the probe set may comprise an isolated nucleic acid that comprises, consists, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 105 and SEQ ID NO: 111 to SEQ ID NO: 308, RNA equivalents thereof, and complements thereof. The isolated nucleic acids may be provided as unmodified probes or may be modified. By way of example and not limitation, the modification may facilitate isolation and/or detection of the probe and a nucleic acid to which it has hybridized, for example, by addition of a ligand and/or detectable labels. In one aspect, the probes may be provided bound to a solid support, such as a plate, tube, bead, microchip, or other solid surface.
Methods of Identifying HPV mRNA
Methods of the present disclosure may be used to detect the presence of a target nucleic acid from samples. Such nucleic acid may be an RNA, and such samples may include, without limitation, a specimen or culture (e.g., cellular, microbiological and viral cultures) including biological and environmental samples. Biological samples may be from a eukaryote, a prokaryote, an archaeon, a virus, an animal, including a human, a plant, a fungus, an excavate, and may be from fluid, solid (e.g., stool) or tissue, cell culture, liquid or solid media, 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, air and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. Particularly preferred are biological samples including, but not limited to, cervical epithelial cells (e.g., a sample obtained from a cervical swab or biopsy), adenoid cells, anal epithelial cells, blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum and semen. The sample may comprise a ribonucleic acid including messenger RNA (mRNA).
The present disclosure provides a method for determining the presence of a target RNA in a sample, wherein the method comprises: a) hybridizing the target RNA with a DNA capture probe having a sequence complementary to the target RNA to form a target RNA:DNA capture probe complex, wherein the DNA capture probe is conjugated to a support; b) separating the target RNA:DNA capture probe complex from unbound RNA (e.g., by washing); c) optionally hybridizing at least one amplification probe to the target RNA:DNA capture probe complex, wherein the at least one amplification probe has a sequence complementary to the target RNA, thereby forming a target RNA:DNA capture/amplification probe complex; d) adding an antibody that recognizes and binds to RNA:DNA hybrids to bind the target RNA:DNA capture/amplification probe complex, thereby forming a target RNA:DNA:antibody complex, wherein the antibody is labeled with a detectable marker; e) detecting the marker on said antibody, wherein the detecting indicates the presence of the target ribonucleic acid; and f) comparing the detection results with results produced from a different combination of amplification probes wherein the comparing indicates the particular RNA splice-form present.
The present disclosure provides a method for determining the presence of a target RNA in a sample, wherein the method comprises: a) hybridizing the target RNA with a DNA capture probe having a sequence complementary to the target RNA to form a target RNA:DNA capture probe complex, wherein the DNA capture probe is conjugated to a support; b) separating the target RNA:DNA capture probe complex from unbound RNA; c) optionally hybridizing at least one amplification probe to the target RNA:DNA capture probe complex, wherein the at least one amplification probe has a sequence complementary to the target RNA, thereby forming a target RNA:DNA capture/amplification probe complex; d) adding an antibody that recognizes and binds to RNA:DNA hybrids to bind the target RNA:DNA capture/amplification probe complex, thereby forming a target RNA:DNA:antibody complex; e) adding a second antibody that recognizes and binds the first antibody, wherein the second antibody is labeled with a detectable marker; f) detecting the marker on the second antibody, wherein the detecting indicates the presence of the target ribonucleic acid; and g) comparing the detection results with results produced from a different combination of amplification probes wherein the comparing indicates the particular RNA splice-form present.
The present disclosure also provides a method of detecting the presence of a ribonucleic acid (RNA) splice form in a sample, wherein the method comprises a) hybridizing the target RNA with a DNA capture probe having a sequence complementary to the target RNA under conditions that allow the probe and the target ribonucleic acid to hybridize, thereby forming a target RNA:DNA capture probe complex; b) adding a first antibody that recognizes and binds to RNA:DNA hybrids to bind the target RNA:DNA capture probe complex, thereby forming a target RNA:DNA capture probe:antibody complex, wherein the first antibody is conjugated to a support; c) separating the target RNA:DNA capture probe:antibody complex from unbound RNA; d) hybridizing at least one amplification probe to the target RNA:DNA capture probe:antibody complex, wherein the at least one amplification probe has a sequence complementary to the target RNA and is added in a combination that will cover specific target RNA regions, thereby forming a target RNA:DNA:antibody complex; e) adding a second antibody that recognizes and binds to RNA:DNA duplexes to bind the target RNA:DNA:antibody complex, to form a target RNA:DNA:antibodies complex, wherein the second antibody is labeled with a detectable marker; f) detecting the marker on said second antibody, wherein the detecting indicates the presence of the target RNA; and g) comparing the detection results with results produced from a different combination of amplification probes wherein the comparing indicates the particular RNA splice-form present.
The present disclosure also provides a method of detecting the presence of a ribonucleic acid (RNA) splice form in a sample, wherein the method comprises a) hybridizing the target RNA with a DNA capture probe having a sequence complementary to the target RNA under conditions that allow the probe and the target ribonucleic acid to hybridize, thereby forming a target RNA:DNA capture probe complex; b) adding a first antibody that recognizes and binds to RNA:DNA hybrids to bind the target RNA:DNA capture probe complex, thereby forming a target RNA:DNA capture probe:antibody complex, wherein the first antibody is conjugated to a support; c) separating the target RNA:DNA capture probe:antibody complex from unbound RNA; d) hybridizing at least one amplification probe to the target RNA:DNA capture probe:antibody complex, wherein the at least one amplification probe has a sequence complementary to the target RNA and is added in a combination that will cover specific target RNA regions, thereby forming a target RNA:DNA:antibody complex; e) adding a second antibody that recognizes and binds to RNA:DNA duplexes to bind the target RNA:DNA:antibody complex, to form a target RNA:DNA:antibodies complex; f) separating the target RNA:DNA:antibodies complex from unbound second antibody; g) adding a third antibody labeled with a detectable marker wherein the third antibody recognizes and binds to the second and/or first antibody; h) detecting the marker on the third antibody, wherein the detecting indicates the presence of the target RNA; and i) comparing the detection results with results produced from a different combination of at least one amplification probe wherein the comparing indicates the RNA splice-form present.
RNA is often transcribed from different promoters and spliced, thereby generating multiple forms that include the coding regions for different genes. It is important to characterize these multiple spliced forms of RNA for fundamental research and for applications where the detection of specific mRNA isoforms is critical.
One application of the present disclosure is the detection and characterization of mRNA expression in human papillomavirus (HPV). Carcinoma of the cervix has been shown to be associated with the presence of high-risk HPV types; from about 13 to about 18 high-risk types are currently identified. The HPV DNA test can identify high-risk HPV types, but is a poor predictor for the progression of the disease in pre-cancerous clinical specimens. Thus, additional methods and markers are needed to improve the predictive value of HPV tests. The characterization of mRNA for the presence of the E6/7 oncogene and other mRNAs, as provided by the present disclosure, will allow an accurate and reliable method that determines the ratio of expression of these oncogenes versus other viral genes. The ratio of E6/E7 to E2, E4, and/or L1 mRNA may be a better predictor for the progression of precancerous cervical lesions (see, e.g., U.S. Pat. No. 6,355,424, incorporated by reference herein). Hybrid capture technology is a linear signal amplification method. Thus, the instant disclosure provides valuable methods for guiding therapeutic strategy, while minimizing the number of patients requiring colposcopy. The instant disclosure provides methods of using mixtures of short oligonucleotides capable of hybridizing to the different lengths/genes of RNA (and mRNA in particular) in order to characterize splice forms.
Target Nucleic Acids
In one aspect, the target ribonucleic acid to be detected may be mRNA, ribosomal RNA, nucleolar RNA, transfer RNA, viral RNA, heterogeneous nuclear RNA etc., wherein the one or more polynucleotide probes are DNA probes. The target ribonucleic acids include, without limitation, nucleic acids found in specimens or cultures (e.g., cellular, microbiological and viral cultures) including biological and environmental samples. The target ribonucleic 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. Target ribonucleic 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. Particularly preferred are target nucleic acids found in biological samples including, but not limited to cervical samples (e.g., a sample obtained from a cervical swab), adenoid cells, anal epithelial cells, blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, urine and semen.
In other aspects, the target ribonucleic acids are from virus, bacteria, mycobacteria or plasmodia, for example, without intending to be limited thereby, cytomegalovirus (CMV), Herpesviridae, human immunodeficiency virus (HIV), Chlamydia spp., Neisseria spp. (e.g., N. gonorrhea), Staphylococcus aureus, mycobacteria (e.g., Mycobacterium tuberculosis), SARS coronavirus (SARS-CoV), or Orthomixoviridae (e.g., influenza viruses).
In one aspect, the target ribonucleic acids are human papillomavirus (HPV) and include genetic variants of HPV. A variant includes polymorphisms, mutants, derivatives, modified, altered, or the like forms of the target nucleic acid. In one aspect, the target nucleic acid is an HPV nucleic acid. In another aspect, the HPV nucleic acid is HPV DNA of a high risk HPV type. In another aspect the target nucleic acids are high risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 66, 73, and 82.
The RNA may be isolated and prepared for hybridization by a variety of methods and reagents including (but not limited to) guanidinium thiocyanate-phenol-chloroform extraction (e.g., with TRIzol® reagent, also known as TRI Reagent), hypotonic lysis, and carboxyl (COOH) bead capture. The principle of RNA isolation is based on cell/tissue lysis, followed by extraction, precipitation, and washing. While very effective, these techniques require a high level of technical precision and are not candidates for automation. Other RNA preparation methods do not completely eliminate DNA and other potential contaminants, require expensive enzymes, and require many sometimes time-consuming—washing steps. The challenge is to develop a method for mRNA detection that reduces many of the current challenges and can provide rapid information about expression of specific genes. Two primary sample preparation methods have been devised for the present disclosure: hypotonic cell lysis; and carboxyl bead capture. RNA isolated using TRIzol® or QIAGEN resin technology (for example, QIAGEN RNeasy Plus Mini Kit) can also be used in this assay.
In certain aspects, the biological sample is comprised of cervical cells, especially human cervical cells. The sample can be collected with any method or device known in the art, including a chemically inert collection device such as a Dacron® (poly(ethylene terephthalate)) tipped swab. Other acceptable collection devices may be used including, but not limited, to cotton swab, cervical brush, flocked swab (a swab shaped like a Dacron® swab but made with nylon fibers enabling collection of more cells and easier release of cells), cervical broom, mini broom, lavage, or any collection device often used in PAP smear testing (Papanicolaou's test). The cervical cells may also be part of a biopsy specimen.
Sample Preparation
The use of TRIzol® to isolate RNA, as well as other known methods for RNA isolation, may be employed in methods of the present disclosure. Sample preparation by hypotonic lysis of the cell pellet reduces the release of endogenous RNA:DNA hybrids that may interfere with assay detection step, and this is a preferable RNA isolation method. In this sample preparation method, cells are pelleted via centrifuge, the supernatant is removed, and the pellet is resuspended and the cells lysed. After lysis, the cellular debris is pelleted and the supernatant (containing RNA) collected. Reducing the stringency of lysis (as measured by salt and detergent concentrations in a buffer) reduces the clinical background produced from pools of methanol-based cervical specimens (FIGS. 5 & 6). The signal:noise ratios are also higher and the variability in background between pools and in interference is lower. Other studies have shown that hypotonic lysis works by rupturing the cellular membrane because of differences in tonicity between the cell and the milieu, making the cell permeable to macromolecules. Thus, RNA in the cell is released from the cell into the solution, whereas contaminants to the assay (such as endogenous RNA:DNA hybrids) will remain in the insoluble cell debris. This method may be useful in cases where the amount of RNA in a specimen is limited because increasing the amount of specimen does not lead to an increase in background.
Another method of sample preparation uses magnetic carboxyl (COOH) beads that can be added directly to a biological sample to concentrate cells for DNA isolation. Cells in the sample are attracted to the beads via hydrophobic interactions. After using a magnetic rack to pellet the beads, the supernatant can be removed and the cells lysed. Non-magnetic COOH beads or other adsorptive particles could also be used, substituting centrifugation for pelleting via a magnetic rack. After the lysis (which usually occurs at 65° C. for 15 min) the beads are again pelleted and the remaining supernatant may be used directly in methods of the present disclosure. While decreasing lysis stringency again reduces background in this method, water alone is not enough to release the RNA from the cells. As such, it is preferable to use a lysis buffer comprising about 1 M guanidine thiocyanate and about 0.7% detergent for all sample preparation methods of the present disclosure (see, e.g., FIGS. 5 & 6).
Hybridization/Capture—Capture Probes
After the sample is prepared and target RNA is released, it is contacted with at least one polynucleotide DNA capture probe under a condition sufficient for the at least one polynucleotide probe to hybridize to the target RNA in the sample to form a double-stranded nucleic acid hybrid. The DNA capture probes may be full length, truncated, or synthetic DNA. The DNA capture probes are sequence specific for the target RNA. DNA capture probes are ideally about 25 to 35 bases long and may be complementary to any region of the target RNA. The DNA capture probes may range from about 15 to about 200 bases in length. In other aspects, the capture probe may be not more than 100 or not more than 50 nucleotides in length. In yet other aspects, the capture probes may be: 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, 45 to 100, or 50 to 100 bases in length.
By way of example and not limitation, the capture probe may comprise, consist essentially of, or consist of at least one nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 20. In a further aspect, the capture probe comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 20. In one aspect, a capture probe set specific for HPV 16 is provided, comprising at least one capture probe selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10. In one aspect, a capture probe set specific for HPV 18 is provided, comprising at least one capture probe selected from the group consisting of SEQ ID NO: 11 to SEQ ID NO: 20.
The DNA capture probes can be bound to a support. “Bound” includes but is not limited to chemically attached, covalently bound, and covalently linked. Multiple DNA capture probes, and multiple different DNA capture probes may be bound to the same support (e.g., the same magnetic bead), as shown schematically in FIG. 3. Only 3-5 different capture probes are required for optimal results (see FIG. 4), thus providing a great deal of flexibility to allow these probes to be sequence-specific and not fall in regions that may be spliced out in some variants. In one aspect, the sequence-specific DNA capture probes are biotinylated and have been bound by conjugation to magnetic streptavidin beads. A capture probe may isolate a particular spliceform if it comprises a single oligo that bridges a splicesite.
Supports include, but are not limited to beads, magnetic beads, columns, plates, filter paper, polydimethylsiloxane (PDMS), and dipsticks. 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 capture probes or antibodies. Magnetic 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 hold the beads in place. Beads that are small and have a high surface area are preferable, such as beads about 1 μm in diameter. In certain aspects, the support comprises a modified magnetic bead, that is coated or has attached thereto a DNA capture probe complementary and specific to the target mRNA. A magnetic field is used to separate the double-stranded nucleic acid/magnetic bead complex from non-bound ribonucleic acid. In certain aspects, the support comprises a modified magnetic bead, wherein the magnetic beads are modified by coating the beads with a first antibody immunospecific for double-stranded hybrid nucleic acids. A magnetic field is used to separate the nucleic acid hybrid/antibody/magnetic bead complex from unbound ribonucleic acid. Other beads that employ charge switching or silica capture (as opposed to magnetic fields) may be used as well. In another aspect, magnetic beads with detection capacity (such as magnetic Lumonex beads) may capture and detect specific spliceforms.
Following capture of the target RNA or the target RNA:DNA hybrid as described above, the captured target RNA or RNA:DNA hybrid may be separated from the rest of the sample by application of a magnetic field (in the case of magnetic beads), and washing away of non-captured nucleic acids. Washing away unwanted interfering substances may be accomplished with buffers containing salt and or detergent that are used at various temperatures. When using supports other than magnetic beads, alternative methods of separating captured hybrid from the rest of the sample are conducted, including but not limited to, washing. Enzymatic processes, such as dnase for double-stranded DNA or RNA:DNA may be used to facilitate isolation of target RNA.
Hybridization/Capture—Amplification Probes
After the wash step to ensure that only the target remains, signal amplification DNA probes are hybridized to the target mRNA, wherein the signal amplification probes are unlabeled DNA probes complementary and/or specific to the target mRNA. The amplification probe need not be specific to the target nucleic acid. For example, the DNA amplification probe may be able to bind other nucleic acids other than the designed target. The DNA signal amplification probes complementary to the mRNA regions are designed and combined in mixtures that will cover specific genes. By extending and varying the coverage, one can determine which genes are present and the particular splice forms of the RNA. “Coverage” is defined as the extent or length of target sequence which is flanked by the complementary signal probes. The signal amplification probes are roughly 40 bases in length, but because they are designed around the capture probes, some may be more or less than 40 bases. Signal amplification probes may be about 15 to about 200 bases in length. In yet other aspects, the signal amplification probes may be: 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, 45 to 100, or 50 to 100 bases in length. Increasing coverage (i.e., hybridizing more signal probes to complementary regions of the target RNA) will lead to an increase in signal. Therefore, it is preferable to use more probes to obtain an amplified signal. The limit of detection depends, in part, on the length of the target nucleic acid (i.e., the target gene).
By way of example and not limitation, the amplification probe may comprise, consist essentially of, or consist of at least one nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 105. In a further aspect, the amplification probe comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 105. In one aspect, an amplification probe set specific for HPV 16 is provided, comprising at least one amplification probe selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 62. In one aspect, an amplification probe set specific for HPV 18 is provided, comprising at least one amplification probe selected from the group consisting of SEQ ID NO: 63 to SEQ ID NO: 105.
Amplification signal probes are added in combinations which would extend over the genetic sequence of known RNA splice-forms. The combination of signal amplification probes will determine the extent of coverage on the target mRNA and hence, signal output. Comparison of the resulting signal output from different combinations of amplification probes will indicate the presence of particular mRNA splice-form variants. In this way, this method is a “molecular ruler” in that the signal output is dependent on the splice form present. For example, capture probe 3 is expected to hybridize with E6/7 target mRNA, but not with E1, E2, E4, E5, L1, or L2 (see, e.g., TABLE 3 and FIG. 12). Signal amplification probes 1 and 6, used after hybridization with capture probe 3, will generate a strong signal from the spliced E6/7 form, and a weak signal from the spliced/integrated E6/7 form. By varying the combinations and numbers of capture probes and amplification probes, the signal output provides information about which viral genes are being expressed (e.g., the ratio thereof), as well as which splice forms of those genes are expressed. Such information, coupled with clinical and experimental data, is expected to provide a better predictor for progression of precancerous cervical lesions.
The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining whether cells are infected with a pathogen, and the state of disease progression.
The present disclosure provides a method of determining lengths of gene transcripts for known and unknown splice form variants. A reliable and robust method for measuring the expression of alternatively spliced transcripts is an important step in investigating the significance of each variant. So far, accurate quantification of splice variants, such as Northern blotting, RT-PCR and real time RT-PCR, has been laborious and difficult due to the intrinsic limitations of conventional methods. The present disclosure provides methods of determining the presence of splice form variants. For example, the question of whether an early HPV transcript (for example HPV E6*I) bears late-gene sequences may be determined by capturing the transcript with capture probes complimentary to the early region, then detecting with amplification probes that are complementary to the late region; resulting signal may indicate the presence of late regions on early gene transcripts. Furthermore, by providing a combination of degenerate signal amplification probes that would cover predicted splice form sequences, the presence of a splice variant could be determined. Furthermore, the absence of a region may be indicated by lack of capture by select DNA probes.
The resulting hybrids are captured/detected using molecules that recognize RNA:DNA hybrids. 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 oligonucleotide or peptide molecules that bind to a particular target molecule. They are often created by selecting them from large pools of random sequences, although naturally-occurring aptamers (e.g., riboswitch aptamers) are known.
Hybridization/Capture—Anti-Hybrid Antibody
In one aspect the molecule specific for the double stranded nucleic acid hybrid is an antibody (“anti-hybrid antibody”). The hybrids are incubated with the anti-hybrid antibody for a sufficient amount of time to allow binding to the double-stranded nucleic acid hybrids. The anti-hybrid antibody may be monoclonal or polyclonal. In a most preferred aspect the antibody is monoclonal.
In another aspect, the first antibody is bound to a support. In this aspect, after the sample is prepared and RNA is released, it is contacted with at least one polynucleotide DNA capture probe under conditions sufficient for the at least one polynucleotide probe to hybridize to the target RNA in the sample to form a double-stranded nucleic acid hybrid. The target RNA, in the form of a target RNA:DNA capture probe complex is separated from unbound RNA by washing. After the wash step to ensure that the only RNA remaining is target RNA, signal amplification DNA probes are hybridized to the target RNA, wherein the signal amplification probes are unlabeled DNA probes that are complementary and/or specific to the target RNA. The hybridization of capture and amplification probes to the target RNA creates double stranded nucleic acid hybrids. The resulting hybrids are detected using molecules that recognize RNA:DNA hybrids. In a preferred aspect the molecule specific for the double stranded nucleic acid hybrid is an antibody (“anti-hybrid antibody”). The hybrids are incubated with the anti-hybrid antibody for a sufficient amount of time to allow binding to the double-stranded nucleic acid hybrid regions. The anti-hybrid antibody is conjugated to a support and binding to the RNA:DNA hybrids forms an RNA:DNA hybrid:antibody complex. The complex is separated from unbound antibody. In applications where the support is a magnetic bead, a magnetic field is used to separate out any unbound antibody.
Detection
After unbound anti-hybrid antibody is removed, a second antibody is added, wherein the second antibody is labeled with a detectable marker and recognizes and binds to the first antibody. The label present on the second antibody is detected to thus indicate the presence of the target ribonucleic acid. 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).
For example, antibodies conjugated with at least one alkaline phosphatase molecule can be detected by chemiluminescence with a reagent such as a Lumi-PhosTM 530 reagent (Lumigen, Detroit, Mich.) or DR2 (Applied Biosystems, Foster City, Calif.) using a detector such as an E/LuminaTM luminometer (Source Scientific Systems, Inc., Garden Grove, Calif.), an Optocomp ITM Luminometer (MGM Instruments, Hamden, Conn.), or the like. As described herein, detection of the label on the second antibody is indicative of the presence of one or more of the target ribonucleic acids in the sample that are complementary to the one or more probes. Following washing, the sample is suspended in a detection buffer that for example, contains the substrate for the label on the second antibody.
Anti-hybrid antibodies can be used and/or coupled to magnetic beads and/or immobilized on a support in the present assay as described below. In a preferred aspect, the antibodies used for capture and detection of the target nucleic acid are monoclonal antibodies. The first and second antibodies may be the same for capture and detection (i.e., produced by the same hybrid myeloma cell line) or may be from different and produced by different hybrid myeloma cell lines. In a most preferred aspect, the first and second monoclonal antibodies used for capture and/or detection are the same and are specific for RNA/DNA hybrids. Also included are immunofragments or derivatives of antibodies specific for double-stranded hybrids, where such fragments or derivatives contain binding regions of the antibody.
For example, a monoclonal RNA:DNA hybrid antibody derived from myeloma cells fused to spleen cells that are immunized with an RNA:DNA hybrid can be used. The hybrid-specific antibody can be purified by affinity purification against RNA:DNA hybrids immobilized on a solid support, for example as described in Kitawaga et al., Mol. Immunology, 19:413 (1982); and U.S. Pat. No. 4,732,847, each of which is incorporated herein by reference.
Other suitable methods of producing or isolating antibodies, including human or artificial antibodies, can be used, including, for example, methods that select recombinant antibody (e.g., single chain Fv or Fab, or other fragments thereof) from a library, or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); and U.S. Pat. Nos. 5,545,806 and 5,545,807).
In yet another aspect, the present disclosure provides kits that allow for the detection of ribonucleic acids in a biological sample or a sample containing nucleic acids. In a preferred aspect, the kit comprises a) a DNA capture probe conjugated to a magnetic bead; b) a DNA amplification probe; c) a first anti-hybrid antibody; d) a detection reagent comprising a second antibody, wherein the second antibody binds the first antibody and is detectably labeled; e) a detergent-based wash buffer and; f) a second detection reagent comprising a substrate for the label on the second antibody. A preferred detergent-based wash buffer is 40 mM Tris-HCl, 100 mM NaCl, 0.5% Triton X-100.
In certain aspects, detection methods of the present disclosure detect RNA by first capturing the target onto complementary biotinylated DNA probes that are conjugated to magnetic streptavidin beads. This probe-bead complex may be preconjugated and is stable at 4° C. for several months. This capture step is preferably performed at 60° C. with constant shaking and allowed to proceed for about 30 minutes (a time sufficient to allow capture). The beads with the captured target are then washed so that any non-target RNA sequences are removed. Because the hybrid capture antibody binds to individual DNA-RNA hybrids, it is preferable to cover the target region with DNA amplification probes to achieve the maximal signal (see FIGS. 1 & 2). Thus, additional probes are then hybridized to the target mRNA. Because only the target is captured at this point, these probes need not be sequence-specific but rather may cover the full length of the gene, excluding regions that are already covered by the biotinylated specific probes. The signal amplification probes are complementary to the mRNA regions and are designed and combined in mixtures that will cover specific genes. By extending and varying the coverage, particular genes and particular splice variants can be determined. These “signal amplification” probes are preferably used at concentration of 4.2 nM. This hybridization also preferably occurs at 60° C. for 30 min at a pH of around 7.8. The hybridization is then followed by detection with the hybrid capture antibody system discussed above (use of anti-hybrid antibody and a second antibody to detect the anti-hybrid antibody).
Method for Determining the Presence, Disruption, or Absence of a Target Nucleic Acid
In another aspect, a method for determining the presence or absence of a target nucleic acid in a sample is provided, said method comprising: (a) treating a first portion of the sample under conditions sufficient to induce the formation of: (α) a first set of DNA:RNA hybrids comprising the target nucleic acid; and (β) a second set of DNA:RNA hybrids comprising a reference nucleic acid; (b) treating a second portion of the sample under conditions sufficient to induce the formation of the second set of DNA:RNA hybrids, but not the first set of DNA:RNA hybrids; (c) generating a detectable signal in the first portion of the sample and the second portion of the sample, wherein the detectable signal has an intensity that correlates with the concentration of DNA:RNA hybrids; and (d) comparing the intensity of the detectable signal in the first portion of the sample and the intensity of the detectable signal in the second portion of the sample, wherein: (α) the target nucleic acid is present in the sample if the intensity of the detectable signal in the first portion of the sample is greater than the intensity of the detectable signal in the second portion of the sample; and (β) the target nucleic acid is absent from the sample if the intensity of the detectable signal in the first portion of the sample is less than or equal to the intensity of the detectable signal in the second portion of the sample.
As used herein, a “portion of a sample” shall refer to the sample separated in any manner. For example, the sample may be separated into equal portions according volume and/or mass. Alternatively, the different portions may be generated by extracting different constituents from the sample. By way of example and not limitation, the “portion of a sample” may refer to a collection of target nucleic acids and reference nucleic acids bound to a support and separated from the rest of the sample. Regardless of how the portion is generated, each portion should comprise roughly equal amounts of reference nucleic acid.
In one exemplary aspect, the first portion of the sample and the second portion of the sample are formed by a method comprising contacting the sample with: (a) a first capture probe specific for the target nucleic acid under stringent conditions, wherein hybridization of the first capture probe to the target nucleic acid generates a first capture complex; and (b) a second capture probe specific for the reference nucleic acid under stringent conditions, wherein hybridization of the second capture probe to the reference nucleic acid generates a second capture complex. The capture complexes may then be bound to the support.
The first and/or second capture probes may be provided covalently bound to the support or may alternatively be adapted to be bound to the support. By way of example and not limitation, the capture probes may be modified with a ligand and the support coated with a moiety capable of binding to the ligand. In such a configuration, the capture probe is bound to the support by virtue of the association between the ligand and the ligand binding moiety. By way of example and not limitation, the ligand may be biotin and the ligand binding moiety is a molecule capable of binding biotin, such as avidin and streptavidin. If desired, the first and second capture probes may have different ligands. In such a case, a first support can be provided capable of binding to both the first and second capture probes, while a second support is provided capable of binding only the second capture probe. In such a manner, a further level of specificity may be added.
In certain other aspects, the capture probe forms a DNA:RNA hybrid with the target and/or reference nucleic acids. In such a configuration, the portions of the sample may be formed by contacting the sample with a support modified by an entity capable of binding to a DNA:RNA hybrid, such as an antibody (or fragment thereof) immunospecific for double-stranded hybrid nucleic acids. The DNA:RNA hybrid formed by the capture probe and the target and/or reference nucleic acid may then be bound to the support and separated from the rest of the sample via binding of the antibody. The antibody may be covalently bound to the support, bound by virtue of a ligand/ligand-binding moiety, or bound by an entity capable of binding to an antibody, such as an Ig-specific antibody, that is coated to the support.
In another aspect, the support is coated with a nucleic acid, referred to herein as an anchor probe. In such a configuration, the capture probes may be designed with sequences capable of hybridizing to at least a portion of the anchor nucleic acid, thereby binding the capture complex to the support. In such a configuration, the capture probe may comprises: (α) a region capable of hybridizing to the target and/or reference nucleic acid under stringent conditions; and (β) a region capable of hybridizing to a sequence of the anchor probe. The anchor probe for each capture probe may be the same, or it may be different. Additionally, each capture probe may comprise a sequence capable of hybridizing to a different sequence of the same anchor probe. The different sequences may be disposed in the same or in different anchor probes.
In another exemplary aspect: (a) the first portion of the sample is formed by a method comprising capturing the first capture complex and the second capture complex to a first support; and (b) the second portion of the sample is formed by a method comprising capturing the second capture complex, but not the first capture complex, to a second support. In such an aspect, the first support may comprise the first and second capture probes covalently bound thereto (or entities capable of capturing the same), while the second support may comprise the second capture probe, but not first capture probes (or entities capable of capturing the same), bound thereto. Alternatively, the first and second supports may be substantially identical. In such a case, the sample should be first separated and then contacted with the appropriate capture probes before being contacted with the respective supports.
In another aspect, the first and second portions of the sample are formed by a method comprising: (a) capturing the first capture complex and the second capture complex to a first support to form the first portion of the sample; and (b) capturing the first capture complex and the second capture complex to a second support to form the second portion of the sample.
Where the portions of the samples are formed by capture to a support, the capture complexes may optionally be washed to remove non-captured nucleic acids. Washing away unwanted interfering substances may be accomplished with buffers containing salt and or detergent that are used at various temperatures.
Once the sample has been separated into the first and second portions and optionally washed, the target and/or reference nucleic acids are detected by forming a first set of DNA:RNA hybrids comprising the target nucleic acid and a second set of DNA:RNA hybrids comprising the reference nucleic acid.
In one aspect, the DNA:RNA hybrids are formed by contacting the portions of the sample with a signal probe capable of forming a DNA:RNA hybrid with the target and/or reference nucleic acid. As used herein, the term “signal probe” refers to any oligo- or polynucleotide capable of hybridizing to the target or reference nucleic acid under stringent conditions to form a DNA:RNA hybrid. The signal probe may be, but is not required to be, specific for the target or reference nucleic acid. For example, the signal probe may be able to bind other nucleic acids other than the designed target. Signal probes preferably are about 15 to about 200 bases in length. In some aspects, the signal probes are designed to be from 35 to 40 nucleotides in length. In other aspect, a signal probe set is provided, comprising a plurality of signal probes capable of hybridizing to distinct regions of the target and/or reference nucleic acids
In one aspect: (a) the first set of DNA:RNA hybrids is formed by a method comprising contacting the sample with a first signal probe capable of hybridizing to the target nucleic acid; and (b) the second set of DNA:RNA hybrids is formed by a method comprising contacting the sample with a second signal probe capable of hybridizing to the reference nucleic acid. In each case, the first portion of the sample should be contacted with both the first and the second signal probes. Where the second portion of the sample comprises both the target and the reference nucleic acids, it should be not be contacted with the first signal probe.
Once the DNA:RNA hybrids are formed, a detectable signal is generated, the intensity of which correlates with the total concentration of DNA:RNA hybrids in the portion of the sample. Where the intensity of the detectable signal is the same or greater in the second portion of the sample as compared to the first portion of the sample, the target nucleic acid is absent. On the other hand, where the intensity of the detectable signal is the less in the second portion of the sample as compared to the first portion of the sample, the target nucleic acid is present.
In some aspects, a plurality of signal probes are designed so as to cover a substantial portion of the target and/or reference nucleic acid. By extending and varying the coverage, one can determine the approximate portion of the target nucleic acid present. Increasing coverage (i.e., hybridizing more signal probes to complementary regions of the target nucleic acid) will lead to an increase in signal. Therefore, it is preferable to use more probes to obtain an amplified signal. The limit of detection depends, in part, on the length of the target nucleic acid (i.e., the target gene). In an aspect, the probe sets comprise probes sufficient to cover at least 70-percent of the target and/or reference nucleic acids. In other aspects, the probe sets comprise sufficient to cover at least at least 75-percent, at least 80-percent, at least 85-percent, at least 90-percent, and at least 95-percent of the target and/or reference nucleic acids. In other aspects, the signal probes of the probe sets are designed to have an average length of from 20 to 50 nucleotides in length.
In an aspect, signal probes are added in combinations which would extend over the genetic sequence of a target mRNA suspected of being truncated or alternately spliced. The combination of signal probes will determine the extent of coverage on the target mRNA and hence, signal output. Comparison of the resulting signal output from different combinations of signal probes will indicate the presence of particular mRNA splice-form variants. In this way, this method is a “molecular ruler” in that the signal output is dependent on the splice form present.
The present disclosure also provides an assay for determining whether a high-risk HPV E2 gene is expressed or disrupted in a host cell, wherein the target nucleic acid is an E2 mRNA and the reference nucleic acid is selected from the group consisting of HPV E1, HPV E6/E7, HPV L1, and HPV L2 mRNAs. Such methods may also be applied to detecting integration of HPV into a host cell genome and/or predicting onset of HPV-related cell transformation and/or cancer, for example cervical cancer. High-risk HPV types include, but are not necessarily limited to, HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82.
In certain aspects, detection methods of the present disclosure detect mRNA by contacting the sample with a biotinylated DNA capture probe complementary to the target nucleic acid and a biotinylated DNA capture probe complementary to the reference nucleic acid, wherein the capture probes are conjugated to magnetic streptavidin beads. The probe-bead complexes may be preconjugated and are stable at 4° C. for several months. This capture step is preferably performed at 60° C. with constant shaking and allowed to proceed for about 30 minutes (a time sufficient to allow capture). The beads with the captured target are then washed so that any non-target/reference RNA sequences are removed. The bead captured targets/reference nucleic acid complexes are then separated into multiple equal portions. Each portion of the sample is then contacted with DNA signal probes sufficient to cover a significant portion of the reference mRNA. The portions are also contacted with DNA signal probes sufficient to cover a progressively increasing portion of the target mRNA. At least one portion should not be contacted with signal probes capable of hybridizing to target mRNA. Because only the target and/or reference mRNA are captured at this point, these probes need not be sequence-specific but rather may cover the full length of the mRNA, excluding regions that are already covered by the biotinylated specific probes. These signal probes are preferably used at concentration of around 4.2 nM. This hybridization also preferably occurs at 60° C. for 30 min at a pH of around 7.8. The hybridization is then followed by detection with the hybrid capture antibody system discussed above (use of anti-hybrid antibody and a second antibody to detect the anti-hybrid antibody).
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.
Example 1
Sample Preparation Via Hypotonic Lysis of Cell Pellet
Endogenous hybrids present a unique challenge to detection assays because they will be detected by the hybrid capture antibody. Thus, sample preparation preferably inactivates the background of endogenouse hybrids by preventing them from adding to signal by sequestration, binding, or degradation. Hypotonic lysis relies on the former strategy. In this method, cells are pelleted via centrifuge, the supernatant is removed, and the pellet is lysed. As is shown in FIG. 6, reducing the stringency of lysis by varying salt and detergent concentrations in a buffer reduces the clinical background produced from pools of methanol-based cervical specimens. The signal:noise ratios are also higher and the variability in background between pools and in interference is lower (TABLE 2). Other studies have shown that hypotonic lysis works by rupturing the cellular membrane because of differences in cellular tonicity compared to the milieu, making the cells permeable to more soluble mRNA, but less soluble to endogenous hybrids and nuclear DNA. Thus, RNA in the cell is released from the cell into solution, whereas contaminants to the assay such as hybrids will remain with the insoluble cell debris. This method may be useful in cases where the amount of RNA in a specimen is limited because increasing the amount of specimen does not lead to an increase in background (FIG. 7). Using a model of spiking HPV positive cells into pools of negative cervical specimens, hypotonic lysis followed by detection methods of the present disclosure can detect HPV E6/7 RNA from just 1000 cells (FIG. 8).
Example 2
Sample Preparation Via Magnetic Carboxyl Beads
Another sample preparation method that has been characterized for use in the methods of the present disclosure uses magnetic carboxyl modified (COOH) beads that can be added directly to a biological sample (e.g., Sera-Mag® Magnetic Carboxylate-Modified Particles; Thermo Fisher Scientific, Inc.). Cells in the sample are attracted to the beads via hydrophobic interactions. After using a magnetic rack to pellet the beads, the supernatant can be removed and the cells lysed. After lysis, the beads are again pelleted and the remaining supernatant is transferred for use in methods of the present disclosure. While decreasing lysis stringency again reduces background in this method (see TABLE 1), water alone is insufficient to release RNA from the cells. Figures in Table 1 represent percents of a 2% solution, not final solutions. Rather, a preferred lysis buffer is about 1 M guanidine thiocyanate and about 0.7% detergent (see FIG. 9), as it supports both lysis and hybridization. Stronger lysis buffer concentrations may be used if it is diluted before the hybridization capture step. As shown in FIG. 10, the capture of cells onto the beads is a biphasic reaction. Carboxyl beads were spiked directly into PreservCyt®-based samples of cervical cells. Approximately 50-60% of all the cells in the samples were attracted to the beads within the first minute of exposure. This process plateaus for at least 15 min, but approximately 30 min after adding the beads at least 95% of the cells have been captured (as measured by counting cells remaining in the supernatant; see FIG. 10). FIG. 11 shows that using methods of the present disclosure results could be obtained using only approximately 1000 HPV positive cells; carboxyl bead cell capture, followed by detection methods of the present disclosure, is more efficient at obtaining mRNA from cells than hypotonic cell lysis followed by detection methods of the present disclosure (see FIG. 11).










TABLE 1







% Lysis Buffer
S/N



















100
1.6



50
3.2



32.5
7.0



25
1.7



0
0.9










Example 3
Effects of Endogenous Hybrids on Assay Background
Endogenous hybrids are often the source of clinical background noise (see FIG. 5). When HPV 16 E6/7 RNA is spiked into clinical pools (with no HPV; KPSTM(−)), the background is high and the signal is masked. However, when the pools are denatured (1.75 M NaOH) and neutralized before the RNA addition, the background is low and the signal is rescued. This reveals the need to eliminate or prevent release of endogenous nucleic acid hybrids before utilizing a detection method that employs antibodies that recognize nucleic acid hybrids.
Example 4
Effect of Lysis Buffer Concentration on Background
Reducing lysis stringency reduces clinical background noise (see FIG. 6). One mL of methanol-based cervical specimens were spun down and the pellets resuspended in buffer at various concentrations (100% buffer=about 3 M guanidine thiocyanate+about 2% detergent), as shown along the x-axis. Pelleted cells were heated for 15 min at 65° C. The final concentration of lysis buffer was then adjusted to 32.5% for the capture of RNA according to methods of the present disclosure. As shown in FIG. 6, the background decreased with decreasing concentrations of lysis buffer. This experiment provides evidence that hypotonic lysis of cells was successful in preventing release of endogenous nucleic acid hybrids. RNA in the cytoplasm is released from the cell whereas contaminants to the assay such as hybrids will remain in the nucleus.
In addition, water lysis gives lower background and variability and higher signal:noise than more stringent lysis (see TABLE 2, below). Values in TABLE 2 are averaged across results from four different clinical pools of cervical specimens. Typically, these pools vary greatly in background.










TABLE 2






Background




Lysis
(RLUs)
Background
S/N Ratio


Condition
1 mL PC- pools
Variability
(104 SiHa Cells)


















Water
71
21.8%
6.6


100% Lysis
652.3
53.2%
4.7


Buffer









Example 5
Hypotonic Lysis of Cell Pellets
FIG. 7 shows that hypotonic lysis of cell pellets ensures that background noise remains stable. Varying amounts of cervical specimens (250 ul-10 ml) were spun down, lysed with water, and subjected to RNA detection assays of the present disclosure. As shown in the graph in FIG. 7, the background does not change significantly regardless of the amount of specimen used.
Example 6
Limit of Detection
The limit of detection for HPV 16 E6/7 RNA from HPV positive cells (SiHa cells) was tested (see FIG. 8). Cells were spiked into 1 mL of a pool of negative cervical specimens to model a clinical sample. After spinning down and being lysed with water and heated, buffer was added to the cells (to a concentration of 32.5% buffer, or about 1M guanidine thiocyanate and about 0.7% detergent) and they were placed in a plate to begin the RNA detection assay of the present disclosure. The results show that using the methods of the present disclosure, as few as 1×103 cells are required for HPV E6/7 RNA detection.
Example 7
Lysing Cells Captured by COOH Beads
Various lysis buffers were compared for the ability to lyse cells captured by COOH beads (see FIG. 9). The results show that water alone is not enough to lyse cells captured by COOH beads. Either HPV negative or HPV positive cells were spiked into 1 mL of a negative cervical pool. After cells were captured by beads and the supernatant removed, varying concentrations of buffer (containing guanidine thiocyanate and detergent) were added to the samples which were then heated for 15 min at 65° C. Buffer concentration was adjusted to a total of 32.5% for RNA detection using methods of the present disclosure. As seen with the spin-down method, background does decrease with decreasing amounts of salt and detergent. However, at least 32.5% buffer (totaling approximately 1 M salt and 0.7% detergent) is required to lyse the cells enough to release RNA.
Example 8
Time Course of Cell Capture by COOH Beads Shows that Capture of Cells onto the Beads is a Biphasic Reaction
A time course of cell capture by COOH beads was conducted (see FIG. 10). Cells were spiked into 1 mL of a negative cervical pool. The baseline number of cells was counted, and at each time point after addition of COOH beads, beads were pelleted for 1.5 min and then the supernatant removed and diluted for counting. Approximately 50% of cells are captured within a minute. Capture then plateaus but at 30 min at least 95% of the cells have been captured. More beads provide slightly more efficient capture.
Example 9
Carboxyl (COOH) Bead Capture is More Efficient than Hypotonic Lysis
HPV 18 positive (HeLa) cells in 1 mL of a pool of negative cervical specimens were prepared with either COOH bead capture or with pelleting and hypotonic lysis. The limit of detection for the carboxyl bead capture method is also approximately 1000 HPV positive cells and the results of the reverse hybrid capture assay show that this method is more efficient for obtaining mRNA from cells (see FIG. 11). While the background is slightly higher when COOH bead capture is used (271 RLUs versus 163 RLUs for hypotonic lysis), both signal:noise and signal—noise (a measure of the total RNA detected) were much higher than when hypotonic lysis is used.
Example 10
Pretreatment Procedure (Hypotonic Lysis) Combined with Detection of Target RNA
The following protocol combines a sample pretreatment procedure (using hypotonic cell lysis) with an RNA detection method of the present disclosure. Spin down cells in tubes for 3 minutes at 1500 relative centrifugal force (RCF). Supernatant was removed and 33.75 μL water was added and pipetted gently to resuspend the pellet. Then, heat for 15 minutes at 65° C. with gentle shaking Next, add 16.25 μL buffer (about 3 M guanidine thiocyanate and about 2% detergent) and transfer 50 μL sample to wells on the plate. Then, add 10 μL preconjugated streptavidin beads with biotinylated capture probes and incubate the plate for 30 minutes at 60° C. with shaking at 1150 revolutions per minute (RPM). Place the plate on a magnetic rack and let the beads pellet for 1.5 min and then decant and blot plate. Wash twice with Sharp Wash buffer (1 M Tris-HCl, 0.6 M NaCl, 0.25% Tween-20); the first wash should be 2 minutes and the second wash should be 5 minutes. After washing, decant and dry plate well by blotting. To each well, add 65 μL signal amplification probes diluted to 4.2 nM in RNA hybridization buffer. Then, incubate the plate for 30 minutes at 60° C. with shaking at 1150 RPM. Place the plate on magnetic rack for 3 min, decant, and dry wells. Add 35 μL Digene Hybrid Capture 2 kit Detection Reagent 1 (alkaline phosphatase-conjugated antibodies to RNA:DNA hybrids in buffered solution with 0.05% (w/v) of sodium azide, and with no RNase) into each well and incubate the plate for 30 minutes at 45° C. Place the plate on the magnetic rack, decant, and blot. Wash the plate five times with buffer comprising 40 mM Tris-HCl, 100 mM NaCl, 0.5% Triton X-100, allow plate to sit 1 minute per wash. Then, decant and dry the wells. Next, add 45 μL Digene Hybrid Capture 2 kit Detection Reagent 2 (CDP-Star® reagent with Emerald IITM, a chemiluminescent substrate) to each well. Protect from light and incubate the plate for 15 minutes at room temperature with shaking at 300 RPM. Read the plate on a luminometer.
Example 11
Pretreatment Procedure (COOH Bead Capture) Combined with Detection of Target RNA
The following protocol combines carboxyl bead capture sample preparation with an RNA detection method of the present disclosure. To each sample, add 8 μL carboxyl (COOH) beads (2 mL well plate) and shake at 800 RPM for 30 minutes at room temperature. Place the plate on a magnetic rack for 2 minutes to pellet beads. Remove supernatant with vacuum and resuspend in 50 μL 32.5% buffer (about 1M guanidine thiocyanate and about 0.7% detergent). Then, shake at 1000 RPM for 15 minutes at 65° C. Place the plate on a magnetic rack, pellet the beads, and transfer supernatant to new wells. Then, add 10 μL preconjugated streptavidin beads with biotinylated capture probes and incubate the plate for 30 minutes at 60° C. with shaking at 1150 RPM. Place the plate on a magnetic rack and let the beads pellet for 1.5 min and then decant and blot plate. Wash twice with Sharp Wash buffer (1 M Tris-HCl, 0.6 M NaCl, 0.25% Tween-20); the first wash should be 2 minutes and the second wash should be 5 minutes. After washing, decant and dry plate well by blotting. To each well, add 65 μL signal amplification probes diluted to 4.2 nM in RNA hybridization buffer. The, incubate the plate for 30 minutes at 60° C. with shaking at 1150 RPM. Place the plate on magnetic rack for 3 min, decant, and dry wells. Add 35 μL Detection Reagent 1 (alkaline phosphatase-conjugated antibodies to RNA:DNA hybrids in buffered solution with 0.05% (w/v) of sodium azide, and with no RNase) into each well and incubate the plate for 30 minutes at 45° C. Place the plate on the magnetic rack, decant, and blot. Wash the plate five times with buffer comprising 40 mM Tris-HCl, 100 mM NaCl, 0.5% Triton X-100, allow plate to sit 1 minute per wash. Then, decant and dry the wells. Next, add 45 μL Detection Reagent 2 (CDP-Star® reagent with Emerald IITM, a chemiluminescent substrate) to each well. Protect from light and incubate the plate for 15 minutes at room temperature with shaking at 300 RPM. Read the plate on a luminometer.
Example 12
Streptavidin Bead-Biotinylated Probe Conjugation
The following protocol provides a method of forming DNA capture probes bound to magnetic beads. Vortex and sonicate Seradyn dsMag streptavidin beads (Seradyn part #3015210301050, Thermo Fisher Scientific, Inc.). Add 5 μL beads to 250 μL bead conjugation buffer (1×PBS; 0.15 M NaCl). Pull down beads on magnetic rack and was twice with bead conjugation wash buffer (above 0.5% Tween-20). Resuspend beads with 45 nM of each DNA capture probe in bead conjugation buffer. Incubate for 30 minutes at 37° C. with shaking at 1150 RPM. Pull down beads and wash three times with bead conjugation wash buffer. Resuspend in 250 μL Blocker buffer (casein-based) from Digene Hybrid Capture 2 to yield 50× beads.
Example 13
Reverse Hybrid Capture Assay
Reverse hybrid capture detects mRNA by first capturing the target RNA onto complementary biotinylated DNA probes that are conjugated to magnetic streptavidin beads. This probe-bead complex may be preconjugated and is stable at 4° C. for several months. This capture step requires 30 min and should occur at 60° C. with constant shaking. The beads with the captured target are then washed so that any non-target RNA sequences are removed. Because the hybrid capture antibody binds to individual DNA-RNA hybrids, it is preferable to cover the target RNA with DNA probes (e.g., DNA capture probe and amplification probes) to achieve the maximal signal (see, e.g., FIGS. 1 & 2). Thus, additional probes are then hybridized to the target mRNA. Because only the target is present at this point (because non-target RNA has been washed away), these probes need not be sequence-specific but rather may cover the full length of the gene, excluding regions that are already covered by the biotinylated DNA probes. These “signal amplification” probes are diluted to a working concentration of 4.2 nM. This hybridization also occurs at 60° C. for 30 min at a pH of around 7.8, preferably with shaking. The hybridization is then followed by detection with the hybrid capture antibody system: exposure to Detection Reagent 1 (alkaline phosphatase-conjugated antibodies to RNA:DNA hybrids in buffered solution with 0.05% (w/v) of sodium azide, and with no RNase) for 30 min at 45° followed by extensive washing and subsequent addition of Detection Reagent 2 (CDP-Start reagent with Emerald IITM, a chemiluminescent substrate) for 15 min at room temperature. The signal is read on a luminometer. This post-analytic portion of the assay takes approximately 2 h 15 min.
Example 14
Effect of Adding Unlabeled Signal Amplification Probe
The signal is relatively low for a RNA target captured with only 3 or 5 biotinylated DNA capture probes and no unlabeled signal probes. The signal is substantially higher when unlabeled probes are hybridized to the target before detection with hybrid-capture antibody and luminescence technology. The reverse hybrid-capture assay is used to detect RNA. In this experiment, a variable number of biotinylated DNA capture probes were conjugated to streptavidin beads (see FIG. 4). The target was the E6/7 gene of HPV 16. The assay was performed with each set of beads with and without the addition of signal amplification probes (one- versus two-step assay, respectively). When no unlabeled DNA probes for signal amplification were added (one-step assay; gray bars), the signal increased with the amount of coverage provided by the biotinylated capture probes. However, when unlabeled DNA probes for signal amplification were added (two-step assay; black bars), the signal was much higher than in the one-step assay when only 1, 3, or 5 capture probes were used. In the two-step assay, optimal signal was achieved with as few as 3 to 5 capture probes.
Example 15
Length of mRNA Transcript Determined by Molecular Ruler Method
The length of HPV transcripts can be “measured” by capture onto magnetic beads and detection with unlabeled oligonucleotides used in order to extend the length of the hybrid region. Signal output will increase with successive addition of amplification signal probes until maximum length is reached, where the signal will plateau. The various HPV transcripts for HPV 16 are shown schematically in FIG. 12. The numbered regions 1 through 7 (FIG. 12) are designated for probe design. For instance, the E6/7 gene transcript can be captured from a sample using the DNA capture probe 3 and the combination of signal amplification probes will determine the signal output. If the variant form present is full length and the combination of amplification probes covers the entire length of the transcript, the signal will be strong. If E6/7 the variant form present is spliced and a subset of signal probes is used (e.g., probes 1 and 6), then the signal output will be somewhat weaker compared to signal from full-length/unspliced E6/7 (see TABLE 3). If the E6/7 variant form is spliced and integrated, it will provide a much weaker signal (see TABLE 3). The stronger signal is indicative of a greater number of targets and a certain disease state. E6/7 spliced integrated variant provides a weaker signal and is indicative of fewer targets captured, and thus less expression of this gene. It is also indicative of a different disease state. TABLE 3 shows the expected signal resulting from the combined use of the listed probes (shown in FIG. 12) from various regions of HPV 16.











TABLE 3





mRNA

Capture
Signal
Signal


Target
Splice Form
Probes
Probes
Output







E6/7
unspliced full length
2
1, 2, 3, 4,
++++++





5, 6, 7



spliced
3
1, 6
++++



spliced/integrated
3
1, 6
+


E2
episomal
5
1, 5, 6
++++++



integrated
5
1, 5, 6
+


L1
spliced
7
4, 6, 7
++++++









Referring again to FIG. 12 and TABLE 3, the signal contributed by non-spliced transcripts hybridizing to capture probe #2 (for example) may be subtracted from the signal generated using other capture probes to determine the degree of signal arising from spliced transcripts alone. The combination of signal amplification probes will determine the extent of coverage on the target mRNA and hence, signal output. Comparison of the signal output resulting from different combinations of amplification probes will indicate the presence of particular mRNA splice form variants. In this way, this method is a “molecular ruler” in that the signal output is dependent upon the splice form present and can indicate progression of disease state.
Example 16
Detection of Elevated Early:Late mRNA Ratio
The methods of the present disclosure enable detection of a ratio of early and late HPV mRNA transcripts, which may be indicative of progressing HPV-related cervical disease. The described assay detected a high early:late mRNA ratio of SiHa cells (cancer cell line) against a background of HPV-positive specimens (FIG. 14). Capture and detection DNA probes were designed to detect early transcripts and late transcripts of HPV. These two assays were performed concurrently on the same samples, and the ratio of the resulting signals indicates the ratio of the early and late HPV transcripts. To mimic specimens comprising a few cancer cells mixed with cells of pre-cancerous lesion, pools of HSIL specimens (high-grade squamous intraepithelial lesion, per Bethesda System for cervical cytology) were spiked with known numbers of SiHa cells (as indicated along the x-axis), and then assayed via the methods of the present disclosure (see, e.g., EXAMPLE 12). As indicated by FIG. 14, a fraction of cells with a high E6/7 mRNA ratio may be detected against a background of cells with a low ratio.
All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.
It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing aspects are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.
Example 17
Use of Nucleic Acids According to the Present Application
Samples and Specimens
Cell lines of SiHa (HTB-35), CaSki (CRL-1550), HeLa (CCL-2) and HCC 1806 (CRL-2335) were obtained from ATCC (Manassas, Va.) and cultured by standard techniques. Residual cervical specimens in liquid-based cytology (LBC) medium (PreservCyt®, Hologics, Ma; 20 ml original volume) were obtained after routine testing from Cytology Services of Maryland. Specimen pools were composed of several of these specimens. These specimens were 5-8 months old and stored at room temperature before use. HPV genotyping of some clinical specimens was done according to Nazarenko et al (2008) to confirm single HPV 16 infection or to confirm the lack of HPV DNA.
RNA Target Isolation
The in vitro transcribed HPV 16 or HPV 18 RNAs for E6 (1-790 nt) and E2 (2755-3852 nt) regions were prepared with standard cloning techniques using HPV 16 (SEQ ID NO: 106) or HPV 18 (SEQ ID NO: 107) as a template. RNA was prepared from samples, cell lines and specimens using either the Rneasy® Plus Mini Kit, or QIAzol lysis reagent (QIAGEN, Valencia, Ca). For QIAzol RNA isolation, the cells preserved in LBC were isolated by centrifugation, the cells were extracted and the precipitated RNA was then resuspended in tris-buffer (pH 7).
Cell Concentration
Some cells preserved in LBC medium (1 ml) from specimens were concentrated in microfuge tubes by adsorption onto carboxyl-modified magnetic beads (8 μl of 5% solids; catalog #65162105050350, Seradyn). The specimen-bead suspension was incubated at 22° C. for 30 min in a rotating microfuge block (1100 rpm, Eppendorf). The cells adsorbed onto beads were pelleted by a magnetic tube holder (Promega, Madison, Wis.). The percent of cells pelleted from mixtures of known cell number was determined by counting the cells in the leftover supernatant using a hemocytometer. The cells were washed with saline and resuspended in lysis buffer then transferred to a 96-well assay plate.
Oligodeoxyribonucleotides
The oligodeoxyribonucleotide (oligo) probes were designed to be specific for HPV 16 (or 18) mRNA targets by using either Blast (NCBI) comparisons. The design of capture probes was adjusted to avoid cross-hybridization with other HPV types. The signal amplification oligos were complimentary to their targets, but not designed to avoid cross-reactivity with other HPV types. Capture and amplification probe sequences are shown below in Table 4. Capture oligos were modified with a 5′ biotin.










TABLE 4







SEQ





ID



Target
Type
NO:
5′-3′ Sequence







HPV 16
Capture
  1
GTTTGCAGCTCTGTGCATAACTGTGGTAACTTTCT


E6-7





 


HPV 16
Capture
  2
CAGTAACTGTTGCTTGCAGTACACACATTCTAATA


E6-7





 


HPV 16
Capture
  3
ACATATATTCATGCAATGTAGGTGTATCTCCATGC


E6-7





 


HPV 16
Capture
  4
AAGGTTACAATATTGTAATGGGCTCTGTCCGGTTC


E6-7





 


HPV 16
Capture
  5
ATTAACAGGTCTTCCAAAGTACGAATGTCTACGTG


E6-7





 


HPV 16
Capture
  6
CAATAGTCTATATGGTCACGTAGGTCTGTACTATC


E2





 


HPV 16
Capture
  7
CAAGGCTAACGTCTTGTAATGTCCACTTTTCATTA


E2





 


HPV 16
Capture
  8
TATAAACCATAATAGTCAACTTGACCCTCTACCAC


E2





 


HPV 16
Capture
  9
TTGGTCACGTTGCCATTCACTATCATATGTAAGTG


E2





 


HPV 16
Capture
 10
CTGATCTTGGTCGCTGGATAGTCGTCTGTGTTTCT


E2





 


HPV 18
Capture
 11
TCATAGTGGTCTATGATTTTGTCCTGCACGCAACT


E6-7





 


HPV 18
Capture
 12
TCCAATCCTCGGTTTTGTATCGACTTTGTGCAAGG


E6-7





 


HPV 18
Capture
 13
TGTGACTTACACAGGTAGCGGTTTTGTCCCATGTT


E6-7





 


HPV 18
Capture
 14
TGGGTTGACAGGTCCACAATGCTGCTTCTCCGCGA


E6-7





 


HPV 18
Capture
 15
CCACCAATATTTGTACACTATCTGGAATTGCAACA


E6-7





 


HPV 18
Capture
 16
ATACACAGGTTATTTCTATGTCTTGCAGTGAAGTG


E2





 


HPV 18
Capture
 17
GCACTGGCCTCTATAGTGCCCAGCTATGTTGTGAA


E2





 


HPV 18
Capture
 18
CATAGAAGGTCAACCGGAATTTCATTTTGGGGCTC


E2





 


HPV 18
Capture
 19
CGGGCTGGTAAATGTTGATGATTAACTCCATCTAT


E2





 


HPV 18
Capture
 20
CAGGGTGTTCAGAAACAGCTGCTGGAATGCTCGAA


E2





 


HPV 16
Amplification
 21
TTTTATACATCCTGTTGGTGCAGTTAAATACACTT


E2





 


HPV 16
Amplification
 22
CCATCAAACTGCACTTCCACTGTATATCCATGTTT


E2





 


HPV 16
Amplification
 23
TCCAGTTTGTATAATGCATTGTATTGCATATGTCT


E2





 


HPV 16
Amplification
 24
AGTTACTGATGCTTCTTCACAAATATATATATGTG


E2





 


HPV 16
Amplification
 25
CTTTATTTTTACTATATTTTTCTGCATCATCTTTAAA


E2





 


HPV 16
Amplification
 26
CATAATATTACCTGACCACCCGCATGAACTTCCCATA


E2





 


HPV 16
Amplification
 27
AGAGGATACTTCGTTGCTGCTAAACACAGATGTAGGA


E2





 


HPV 16
Amplification
 28
CGGGGTGGTTGGCCAAGTGCTGCCTAATAATTTCAGG


E2





 


HPV 16
Amplification
 29
CTGCACAAAATATGTTCGTATTCCTTCATGAACATAA


E2





 


HPV 16
Amplification
 30
TCGGTGCCCAAGGCGACGGCTTTGGTATGGGTCGCGG


E2





 


HPV 16
Amplification
 31
CACACATTTAAACGTTGGCAAAGAGTCTCCAT


E2





 


HPV 16
Amplification
 32
ATTTTCATAATGTGTTAGTATTTTGTCCTGA


E2





 


HPV 16
Amplification
 33
TAGTTTTTGGTATTTTAACTTGAGACAAAAA


E2





 


HPV 16
Amplification
 34
TCATATAGACATAAATCCAGTAGACACTGTAA


E2





 


HPV 16
Amplification
 35
TAATAAATAGCACATTCTAGGCGCATGTGTTTC


E2





 


HPV 16
Amplification
 36
TTAATATGTTTAAATCCCATTTCTCTGGCCTTG


E2





 


HPV 16
Amplification
 37
TTTGATACAGCCAGTGTTGGCACCACTTGGTGG


E2





 


HPV 16
Amplification
 38
AGTTGCAGTTCAATTGCTTGTAATGCTTTATTC


E2





 


HPV 16
Amplification
 39
CTATATTGTGAGTTATATATTGTTTCTAACGTT


E2





 


HPV 16
Amplification
 40
TAGTGGTGTGGCAGGGGTTTCCGGTGTCTGGCT


E2





 


HPV 16
Amplification
 41
TAACAATTGCACTTTTATGTTTTACATTATGTC


E2





 


HPV 16
Amplification
 42
GGAGCACTGTCCACTGAGTCTCTGTGCAACAACT


E2





 


HPV 16
Amplification
 43
TCCTTTGTGTGAGCTGTTAAATGCAGTGAGGATT


E2





 


HPV 16
Amplification
 44
CTATGGGTGTAGTGTTACTATTACAGTTAATCCG


E2





 


HPV 16
Amplification
 45
CATTTTAAAGTATTAGCATCACCTTTTAAATGTA


E2





 


HPV 16
Amplification
 46
CAATGTACAATGCTTTTTAAATCTATATCTTAAA


E2





 


HPV 16
Amplification
 47
CTGTCCAATGCCATGTAGACGACACTGCAGTATA


E2





 


HPV 16
Amplification
 48
ATACTATGCATAAATCCCGAAAAGCAAAGTCATATAC


E6-7





 


HPV 16
Amplification
 49
ATTTATCACATACAGCATATGGATTCCCATCTCTAT


E6-7





 


HPV 16
Amplification
 50
GTCTATACTCACTAATTTTAGAATAAAACTTTAAAC


E6-7





 


HPV 16
Amplification
 51
GTTCTAATGTTGTTCCATACAAACTATAACAATAAT


E6-7





 


HPV 16
Amplification
 52
CTAATTAACAAATCACACAACGGTTTGTTGTATTGCT


E6-7





 


HPV 16
Amplification
 53
CCTGTGGGTCCTGAAACATTGCAGTTCTCTTTTGGTGCAT


E6-7





 


HPV 16
Amplification
 54
TGTGCTTTGTACGCACAACCGAAGCGTAGAGTCACACTTG


E6-7





 


HPV 16
Amplification
 55
TTATGGTTTCTGAGAACAGATGGGGCACACAATTCCTAGT


E6-7





 


HPV 16
Amplification
 56
TTTTCTTCAGGACACAGTGGCTTTTGACAGTTAATACAC


E6-7





 


HPV 16
Amplification
 57
ATATTATGGAATCTTTGCTTTTTGTCCAGATGTCTTTGC


E6-7





 


HPV 16
Amplification
 58
CTGCAACAAGACATACATCGACCGGTCCACCGACCCCTT


E6-7





 


HPV 16
Amplification
 59
ATGATTACAGCTGGGTTTCTCTACGTGTTCTTGATGAT


E6-7





 


HPV 16
Amplification
 60
CTCCTCTGAGCTGTCATTTAATTGCTCATAAC


E6-7





 


HPV 16
Amplification
 61
AGTAGAGATCAGTTGTCTCTGGTTGCAAATCTA


E6-7





 


HPV 16
Amplification
 62
TGCTTGTCCAGCTGGACCATCTATTTCATCCTC


E6-7





 


HPV 18
Amplification
 63
TAAACGTTCCGAAAGGGTTTCCTTCGGTGTCTGCAT


E2





 


HPV 18
Amplification
 64
ATACTGTATTTGGCTGTCTATGTCTTTACTGTCATTT


E2





 


HPV 18
Amplification
 65
AAAGAATATTGCATTTTCCCAACGTATTAGTTGCCA


E2





 


HPV 18
Amplification
 66
GGTGGTTTAATGTCTGTATGCCATGTTCCCTTGCTGC


E2





 


HPV 18
Amplification
 67
CTTTACTTTTTGAAATGTTATAGGCTGGCACCACCT


E2





 


HPV 18
Amplification
 68
CCTTGTAGGGCCATTTGCAGTTCAATAGCTTTATGTG


E2





 


HPV 18
Amplification
 69
GTTCTGTATTCCATAGTTCCTCGCATGTGTCTTGCAGTG


E2





 


HPV 18
Amplification
 70
TTGTACTGTTTGGCCACCTTTTTTAAAGCAGTGAGTAG


E2





 


HPV 18
Amplification
 71
TAGGTCATACAATTGTCTTTGTTGCCATCAAAATATAC


E2





 


HPV 18
Amplification
 72
CCTGCATCAGTCATATAATACACACTGTCCCATGCTACA


E2





 


HPV 18
Amplification
 73
ACGTGTTGTACCCTTCCTTTACATAATACAATCCCC


E2





 


HPV 18
Amplification
 74
ATATTTTTCACATTCACTTTTAAATTCTATATAAA


E2





 


HPV 18
Amplification
 75
TTCCCAAAATGTACTTCCCACGTACCTGTGTTCCC


E2





 


HPV 18
Amplification
 76
TACTGCACATAGAGTCATTACAATCAATTACATTA


E2





 


HPV 18
Amplification
 77
AACAAGCTGAGTAGCGGATACCGTGTCGTCACTGG


E2





 


HPV 18
Amplification
 78
CTGGAATACGGTGAGGGGGTGTGCTGTAGCTGTTT


E2





 


HPV 18
Amplification
 79
GGCCGTAGGTCTTTGCGGTGCCCACGGACACGGTG


E2





 


HPV 18
Amplification
 80
GTCCACAGTGTCCAGGTCGTGTAGCAGCCGACGTCT


E2





 


HPV 18
Amplification
 81
TTTGTTGTTGCCTGTAGGTGTAGCTGCACCGAGAAG


E2





 


HPV 18
Amplification
 82
TATAGGCGTAGTGTTACCACTACAGAGTTTCCGTCT


E2





 


HPV 18
Amplification
 83
ACATTTTAAACTGTTTCTGTCACCTTTTAAATGTAT


E2





 


HPV 18
Amplification
 84
TAGTGGTCGCTATGTTTTCGCAATCTGTACCGTAA


E2





 


HPV 18
Amplification
 85
GCACCTGTCCAATGCCAGGTGGATGATATATCTCTA


E2





 


HPV 18
Amplification
 86
TATGTTACAGTCAGTATTCCTGTTTTTTCATTGCCT


E2





 


HPV 18
Amplification
 87
GTATTTAAAAATTTTGTTCTTTGTGTTTCACTATGG


E2





 


HPV 18
Amplification
 88
GGGTCGCCGTGTTGGATCCTCAAAGCGCGCCAT


E6-7





 


HPV 18
Amplification
 89
TTCAGTTCCGTGCACAGATCAGGTAGCTTGTA


E6-7





 


HPV 18
Amplification
 90
TCTGTAAGTTCCAATACTGTCTTGCAAT


E6-7





 


HPV 18
Amplification
 91
CACCACAAATAAATCTTTAAATGCAAATTCAAATACC


E6-7





 


HPV 18
Amplification
 92
ATTTATGGCATGCAGCATGGGGTATACTGTCTCTATA


E6-7





 


HPV 18
Amplification
 93
GTCTTAATTCTCTAATTCTAGAATAAAAATCTATAC


E6-7





 


HPV 18
Amplification
 94
TTTTCCAATGTGTCTCCATACACAGAGTCTGAATAAT


E6-7





 


HPV 18
Amplification
 95
CCTTATTAATAAATTGTATAACCCAGTGTTAGTTAGT


E6-7





 


HPV 18
Amplification
 96
TGCTGGATTCAACGGTTTCTGGCACCGCAGGCA


E6-7





 


HPV 18
Amplification
 97
ATCGTCGTTTTTCATTAAGGTGTCTAAGTTTTTC


E6-7





 


HPV 18
Amplification
 98
TTGGAGTCGTTCCTGTCGTGCTCGGTTGCAGCACGAATG


E6-7





 


HPV 18
Amplification
 99
ATGCATACTTAATATTATACTTGTGTTTCTCTGCGTCG


E6-7





 


HPV 18
Amplification
100
TAAATGCAATACAATGTCTTGCAATGTTGCCTTAGGTCC


E6-7





 


HPV 18
Amplification
101
TTCATCGTTTTCTTCCTCTGAGTCGCTTAATTGCTCGTGA


E6-7





 


HPV 18
Amplification
102
CAACATTGTGTGACGTTGTGGTTCGGCTCGT


E6-7





 


HPV 18
Amplification
103
AATTCTGGCTTCACACTTACAACACATACA


E6-7





 


HPV 18
Amplification
104
GGTCGTCTGCTGAGCTTTCTACTACTAGCTC


E6-7





 


HPV 18
Amplification
105
TTACTGCTGGGATGCACACCACGGACACACAAAGGA


E6-7









The reverse-transcription, PCR primers and TaqMan probes were designed by PrimerQuest (IDT, Coralville, Iowa) and Beacon Designer (Palo Alto, Calif.). All oligonucleotides were synthesized by IDT (Coralville, Iowa).
Realtime, Reverse-Transcription and PCR (RT-PCR)
One-step RT-PCR was performed using the QuantiTect® 5× Virus Mix (no rox; QIAGEN, Valencia, Calif.), according to vendor protocol. Primer and probe sets were designed for the E6-7 region and the E2 region using software. Realtime, RT-PCR was performed using either a Stratagene MX3000P (Stratagene, LaJolla, Calif.) or Bio-Rad iQTM5 (Bio-Rad, Hercules, Calif.) realtime PCR instrument. RT-PCR volumes were 25 μl. Consensus PCR (Nazarenko et al., 2008) was used to indicate whether a cervical specimen pool contained HPV DNA types.
Hybrid Capture Assay
The RNA isolation occurred in 60 μl of lysis buffer (RLT Plus, QIAGEN Inc) with the addition of 10 μl of magnetic beads (streptavidin-modified, 0.01% solids, 1 μl, Seradyn). Biotinylated, capture oligos were coupled to the magnetic beads using standard procedures. There were five sequence-specific capture probes per target. The target RNA was captured onto these oligo-modified beads during incubation at 60° C. for 30 min with 1100 rpm rotation. This sample was diluted 1:3 with pure water and split into two wells of a 96-well microtiter plate. Amplification DNA probes (4.2 mM each, 33-45 nt) were hybridized to the target RNA in a buffer composed of a 5:8 mixture of Denaturation Reagent: Probe Diluent (QIAGEN Inc). There were 15 amplification probes for the E6-7 target and 27 amplification probes for the E2 target. The resulting hybrids affixed to beads were pelleted using a magnetic plate holder (Ambion). The hybrid-bead complex was washed on the magnetic plate with a saline, detergent-based buffer (pH 7.5). The complex was incubated (45° C., 30 min) with monoclonal Hybrid Capture antibodies conjugated to alkaline phosphatase (DR1; QIAGEN Inc). This complex was then washed with HC2 wash buffer (QIAGEN Inc). The complex was then incubated (22° C., 15 min, rotation 300 rpm) with a chemiluminescent, alkaline phosphatase substrate (DR2, QIAGEN Inc). The signal was measured in relative luminescence units (RLU) using a DML 2000 luminometer (QIAGEN Inc).
Results
Stability of HPV mRNA
The stability of the HPV mRNA in cells that were fixed in LBC medium was determined using a realtime, RT-PCR assay. SiHa cells contain 2 copies of integrated HPV 16 genome (no episomal) and express HPV E6-7 mRNA. Fresh SiHa cells were preserved in pooled LBC cervical specimens that previously contained no HPV as indicated by PCR. These samples were incubated at room temperature for up to 67 days. Two aliquots (1 ml) were removed periodically (3, 13, 26, 42 and 67 days) and the RNA was isolated by QIAzol reagent. The HPV mRNA level was determined using a realtime, RT-PCR (5′-3′; Forward primer GCACCAAAAGAGAACTGCAATGT (SEQ ID NO: 108), reverse primer CATATACCTCACGTCGCAGTAACT (SEQ ID NO: 109), TaqMan probe FAM-CAGGACCCACAGGAGCGACCCAGA-BHQ1 (SEQ ID NO: 110)). Each reaction contained the mRNA from approximately 125,000 SiHa cells. The cycle threshold, a measure of mRNA abundance, of the RT-PCRs was relatively stable up to 42 days and then shifted by approximately 1-2 cycles for the 42 and 67 day aliquots (FIG. 15). This shift may account for a reduction in target mRNA of approximately 3-fold based on theoretical PCR kinetics.
Analytical Performance of the Hybrid Capture mRNA Detection Assay
A schematic diagram for the hybrid-capture assay for mRNA is shown in FIG. 16a. The assay is loosely based on the digene HC2 HPV DNA Test® (QIAGEN Inc), except mRNA is the target and the probes consist of synthetic DNA (not RNA), the alkali denaturation of target is not included, and the formed RNA:DNA hybrids are captured on magnetic beads instead of an ELISA plate. Four hybrid capture assays for HPV mRNAs were designed to be specific to either HPV 16 E6-7 or E2, or HPV 18 E6-7 or E2, by using specific capture probes (Table 4). The specificity of the capture probes was confirmed by the Blast program. Cell pellets or RNA were lysed to release and unwind the RNA. The lysate was split equally into separate wells for detection of either E6-7 or E2 mRNA. Each well received a unique set of sequence-specific, capture probes (five, 35 nt) affixed to magnetic beads. After washing un-bound material, some amplification probes (33-45 nt) were added to complement the entire length of the E6-7 or E2 coding region for each captured mRNA target. These amplification probes were not designed to avoid cross-reactivity with other HPV types. Their function was to provide signal amplification via increased binding of hybrid capture antibody with alkaline phosphatase. The lengths of the formed hybrid targets were approximately 740 bp for the E6-7 and 1500 bp for the E2. The probe loci for hybrid capture probes are indicated in FIG. 16b. 
The hybrid capture assay was first performed for pure, in vitro transcribed HPV RNA targets for HPV 16 E6-7, HPV 16 E2, HPV 18 E6-7 or HPV18 E2. The results for the HPV 16 E6-7 assay are shown in FIG. 17b. The assay detected approximately 1000 copies of RNA per reaction. There was a linear dependence of signal on target input with a dynamic range of 3-4 logs. A similar dependence of signal on target input was detected for the other three RNA targets including HPV 16 E2, HPV 18 E6-7, and HPV 18 E2 transcripts. No signal above background resulted when an HPV 18 RNA target was probed with the HPV 16 specific probes, or visa versa.
In addition to the amount of target, the assay signal depended on length of formed hybrid allowing the assay to be used as a molecular ruler. To demonstrate this, the relative length of the HPV 16 E6-7 in vitro transcribed RNA was measured by the dependence of the signal on the number of adjacent amplification probes used to lengthen the hybrids. Equivalent amounts of HPV 16 E6-7 RNA were captured by magnetic beads (five capture oligos) in several wells. An increasing number of adjacent amplification probe types were added to each separate well. Thus, each well had RNA: DNA hybrids of successively longer length, until some wells contained completely hybridized RNA targets (FIG. 17a). The signals for wells increased until a plateau was reached at the well in which the target was completely hybridized (15 amplification probes added). The further addition of five, non-complementary probes did not increase the assay signal.
The hybrid capture assay detected the HPV 16 mRNA of SiHa cells preserved in a pool of LBC clinical specimens which previously did not contain HPV. The cell concentration procedure using magnetic, carboxyl-coated beads was applied to pellet the SiHa and other cells, as described in methods. Ninety-five percent of the cells were pelleted in 30 min using this procedure; as determined by cell counting with a hemocytometer. The resulting cell pellets were lysed and the lysate was divided equally (by volume) into two wells. HPV 16 E6-7 transcripts were assayed in one well and HPV 16 E2 were assayed in a second well. The SiHa mixture expressed abundant E6-7 transcripts, but not E2 (FIG. 18a). These assays for HPV 16 detected only a negligible signal when HPV 18 mRNA of HeLa cells (1×106 cells) was used as a target (S:N<2; not shown). Cross-reactivity with other HPV types was not tested. The ratio of HPV 16 E6-7 and E2 in SiHa cells may be calculated from this data. The maximum signal for the E2 hybrid was proportionally greater than for E6-7 hybrid due to its increased length. For this reason the E2 signal was divided by a factor of 0.51 when calculating E6-7:E2 ratios for cells and specimens. The HPV 16 E6-7:E2 ratio was 8.2 or higher for SiHa cells depending upon the number of cells in the assay. This method was used to calculate the HPV E6-7:E2 ratio for other cancer cell lines that express HPV transcripts. These include Caski and HeLa, which express HPV 16 and HPV 18, respectively (FIG. 18b). The ratio for the SiHa and HeLa cell lines was relatively higher than for Caski cells.
Experiments were performed to determine the HPV E6-7:E2 ratios in heterogeneous mixtures of cancer cells and non-cancer cells that both express HPV E6-7 and E2 transcripts in un-equal ratios. The SiHa cells, which have a relatively high E6-7:E2 transcript ratio, were added and preserved in a pool of clinical specimens (LBC medium) that was positive for only HPV 16. The HPV 16 E6:E2 ratio of the pooled specimens was approximately 1, with no added SiHa cells. Serial dilutions of SiHa cells were added to 2 ml aliquots of this specimen pool. The sample RNA was isolated by QIAzol extraction. The results of the HPV E6-7 and E2 assays were expressed as a ratio (FIG. 19). The addition of SiHa cells to the HPV-positive pool resulted in an increased E6-7:E2 ratio, with a substantial increase of approximately 4.2-fold upon addition of 100,000 SiHa cells. In comparison, the ratio for SiHa cells alone (33,000 cells) was about 9 (FIG. 18b).
The HPV 16 E6-7:E2 ratio was determined also in a limited number (n=13) of cervical specimens using the hybrid capture assay for HPV 16 E6-7 and E2. The histological diagnoses of the specimens were known and all specimens were confirmed by PCR to include only HPV 16. The specimen RNA was isolated by QIAzol extraction. There was a broad distribution of ratios for all histological grades, but some specimens had a relatively high ratio (FIG. 20).
This hybrid capture assay detected in vitro transcribed RNA with good linearity and dynamic range of approximately 3-4 logs. This analytical performance is similar to that of hybrid capture detection of DNA. There was no cross-reactivity between the HPV 16 and HPV 18 mRNA due to the specificity of the capture oligos. The cross-reactivity of all the various HPV types was not tested. Detection of E6-7 or E2 mRNA from either HPV 16 or HPV 18 was demonstrated by assays in separate wells. HPV E6-7:E2 ratios may be calculated from these separate assays. The use of short DNA probes for target capture and detection allow flexibility for design with various targets. The assays may be designed to detect a single HPV type (typing) in a single well or to detect simultaneously multiple, specific HPV sequences of various types (screening).
Example 18
Method for Determining the Presence or Absence of a Target Nucleic Acid
Examples 18-21 utilize a two hybridization step assay as exemplified in FIG. 2. In the first hybridization step, an RNA is captured by biotinylated DNA probes that have been conjugated to magnetic streptavidin beads. After extraneous RNA has been washed away, a second round of DNA probes that cover the full length of the RNA target is added. While the first set of DNA probes must be specific in order to ensure that only the desired RNA target is captured, the second round of probes does not need to be specific because only the target RNA is present in the wells for this step. The hybrids are then detected by the two-step Hybrid Capture antibody system (Qiagen Gaithersburg, Inc., Gaithersburg, M(D) and the signal is read on a luminometer. This method allows for linear detection of RNA based on both quantity and length. One may apply a “molecular ruler” concept to this assay in which increasing amounts of signal probes may be added to determine, for example, the length of a transcript.
Although the following Examples use RNA, the general concept may be applied to any form of nucleic acid.
Example 18: Materials and Methods
In Vitro Transcribed RNA
In vitro transcribed RNA from HPV 16 RNA for E6/E7 (790 nucleotides) was used in some of the following examples. The RNA was prepared with standard cloning techniques using HPV 16 plasmid as a template (GenBank NC_01526, X05015)
Clinical Samples
Cervical specimens in PRESERVCYTTM media testing positive for high-risk HPV via the Hybrid Capture II test were obtained during 2009. All samples were genotyped using gp+ consensus primers. Representative samples testing positive for HPV 16 were used in this study. Of these, 14 were diagnosed as LSIL and 35 as high-grade cervical interepithelial neoplasia (HSIL). Each sample was analyzed for the integrity of E2 gene expression.
RNA Extraction
RNA was extracted from samples using the QIAZOLTM reagent (Qiagen GmbH, Hilden, Germany). The entire contents of the sample (ranging from 2-16 ml) was centrifuged for 15 min. The cell pellet was resuspended in 3 ml QIAZOLTM and incubated at RT for 5 min to achieve complete lysis. 0.6 ml of chloroform was then added and the samples were shaken vigorously, then incubated again for 2-3 min at RT and centrifuged for 15 min at 12,000×g. The colorless aqueous layer was transferred to a new tube containing 1.5 ml isopropanol and was incubated at RT for 10 min. Another centrifugation at 12,000×g for 10 min at 4° C. was then performed during which a precipitate formed on the side of the tube. The supernatant was removed and the pellet was washed once with 3 ml 75% ethanol. After the pellet was allowed to air-dry for 10 min, it was resuspended in 50 μl molecular biology grade water.
DNA Probes
DNA capture probes for HPV 16 of 35 nucleotides each were designed using BLAST (NCBI) to be specific against other HPV types. These probes were spaced along the HPV gene so that each possible RNA transcript would be captured by a probe. These capture probes were synthesized with a biotin on the 5′ end. Signal probes were then designed to cover the remainder of the HPV 16 gene. Length of these probes varied from 28 to 42 nucleotides and the OligoAnalyzer program (Integrated DNA Technologies, Inc., Coralville, Iowa) was used to achieve optimal thermodynamic stability and consistency. These probes were then pooled in a probe cocktail. A separate probe cocktail, lacking all probes in the E2 region, was also made.










TABLE 5








Position



Region Detected
HPV 16 (GenBank NC 001526)









All early genes
130-164



E6 (unspliced, E6*1, E6*2)
592-626



E1
1803-1837



E2-amino
3014-3048



E2-hinge
3359-3393



E2-carboxyl
3851-3885



L2
4907-4941



L1
6476-6510










DNA probes were used in the reverse hybrid capture HPV 16 E2 disruption assay. Two probe sets were used in the assay, set one included probes spread along the HPV 16 genome and set two was a subset with no probes included for the E2 gene region. The probes are listed in Tables 6 & 7, with the biotinylated capture probes listed in Table 7.











TABLE 6





SEQ





ID


Probe


NO
Name
Sequence 5′-3′ antisense
set


















111
HPV16-129-33
GGTCGCTCCTGTGGGTCCTGAAACATTGCAGTT
1, 2


 


112
HPV16-193-28
CATTCTAATATTATATCATGTATAGTTG
1, 2


 


113
HPV16-225-32
TCACGTCGCAGTAACTGTTGCTTGCAGTACAC
1, 2


 


114
HPV16-262-37
TACTATGCATAAATCCCGAAAAGCAAAGTCATATACC
1, 2


 


115
HPV16-298-36
TTTATCACATACAGCATATGGATTCCCATCTCTATA
1, 2


 


116
HPV16-335-37
GTCTATACTCACTAATTTTAGAATAAAACTTTAAACA
1, 2


 


 51
HPV16-371-36
GTTCTAATGTTGTTCCATACAAACTATAACAATAAT
1, 2


 


 52
HPV16-408-37
CTAATTAACAAATCACACAACGGTTTGTTGTATTGCT
1, 2


 


 56
HPV16-447-39
TTTTCTTCAGGACACAGTGGCTTTTGACAGTTAATACAC
1, 2


 


 57
HPV16-486-39
ATATTATGGAATCTTTGCTTTTTGTCCAGATGTCTTTGC
1, 2


 


 58
HPV16-525-39
CTGCAACAAGACATACATCGACCGGTCCACCGACCCCTT
1, 2


 


 59
HPV16-556-31
CAGCTGGGTTTCTCTACGTGTTCTTGATGAT
1, 2


 


117
HPV16-591-35
TTCATGCAATGTAGGTGTATCTCCATGCATGATTA
1, 2


 


118
HPV16-669-42
ATCCTCCTCCTCTGAGCTGTCATTTAATTGCTCATAACAGTAG
1, 2


 


119
HPV16-700-31
GTTCTGCTTGTCCAGCTGGACCATCTATTTC
1, 2


 


120
HPV16-731-31
AAGGTTACAATATTGTAATGGGCTCTGTCCG
1, 2


 


121
HPV16-762-31
CAACCGAAGCGTAGAGTCACACTTGCAACAA
1, 2


 


122
HPV16-838-41
TGGGGCACACAATTCCTAGTGTGCCCATTAACAGGTCTTCC
1, 2


 


123
HPV16-880-42
CTGCAGGATCAGCCATGGTAGATTATGGTTTCTGAGAACAGA
1, 2


 


124
HPV16-915-35
TCCATTACATCCCGTACCCTCTTCCCCATTGGTAC
1, 2


 


125
HPV16-950-35
GTTTTTTTTTCCACTACAGCCTCTACATAAAACCA
1, 2


 


126
HPV16-985-35
CATTTTCGTTCTCGTCATCTGATATAGCATCCCCT
1, 2


 


127
HPV16-1020-35
TATAAAATCTACCAAATCTTCACCTGTATCACTGT
1, 2


 


128
HPV16-1055-35
GTTTCTGCCTGTGTTAAATAATCATTATCATTTAC
1, 2


 


129
HPV16-1090-35
CTTCCTGTGCAGTAAACAACGCATGTGCTGTCTCT
1, 2


 


130
HPV16-1125-35
TTTTAGAACCTGTACTGCATCTCTATGTTGTTTTG
1, 2


 


131
HPV16-1160-35
ACTAATATCACTAAGTGGACTACCAAATACTTTCG
1, 2


 


132
HPV16-1195-35
TTTAATCTAGGACTAATATTATTGTCTACACATCC
1, 2


 


133
HPV16-1230-35
TTGCAGCTCTACTTTGTTTTTCTATACATATAGCT
1, 2


 


134
HPV16-1265-35
ATACCCGCTGTCTTCGCTTTCAAATAATCTCCTTT
1, 2


 


135
HPV16-1300-35
TGTAACATCTGCTGAGTTTCCACTTCAGTATTGCC
1, 2


 


136
HPV16-1335-35
TACATGGTGTTTCAGTCTCATGGCGCCCTTCTACC
1, 2


 


137
HPV16-1370-35
ACTGCAACCACCCCCACTTCCACCACTATACTGAC
1, 2


 


138
HPV16-1405-35
CTAACACCCTCTCCCCCACTTCCACTACTGTACTG
1, 2


 


139
HPV16-1440-35
TTGTAAGTGGTGTTTGGCATATAGTGTGTCTTTCA
1, 2


 


140
HPV16-1475-35
CTTTGCATTACTAGTTTTTAGTACATTTAAAATAT
1, 2


 


141
HPV16-1510-35
CCGTATAACTCTTTAAATTTTGCTAACATTGCTGC
1, 2


 


142
HPV16-1545-35
TTTTAAATGGTCTTACTAATTCTGAAAAACTCACC
1, 2


 


143
HPV16-1580-35
AGCAATACACCAATCGCAACACGTTGATTTATTAC
1, 2


 


144
HPV16-1615-35
ATACTGTCAGCTATACTGGGTGTAAGTCCAAATGC
1, 2


 


145
HPV16-1650-35
GTAAATATAAACAATATTGTTGTAATAGTGTTTTT
1, 2


 


146
HPV16-1685-35
AACCATTCCCCATGAACATGCTAAACTTTGAATGT
1, 2


 


147
HPV16-1724-39
TCTATTTTTTCCACATTTATATCTTACTAATAGTAACAC
1, 2


 


148
HPV16-1763-39
CACACATAATAGTTTAGACAGCAATTTTTCAATTGTTAC
1, 2


 


149
HPV16-1802-39
ACGCAATTTTGGAGGCTCTATCATCATACACATTGGAGA
1, 2


 


150
HPV16-1872-35
GCGTGTCTCCATACACTTCACTAATATTTGATATA
1, 2


 


151
HPV16-1907-35
ATGTTGTAATACTGTTTGTCTTTGTATCCATTCTG
1, 2


 


152
HPV16-1942-35
ATCTGTGATAATTCAAATGTACAATCATTAAAACT
1, 2


 


153
HPV16-1977-35
CGTCTACTATGTCATTATCGTAGGCCCATTGTACC
1, 2


 


154
HPV16-2012-35
TGCCAATTGTGCATATTTATATGCAATTTCACTAT
1, 2


 


155
HPV16-2047-35
CTTTTTAGAAAGGCACTTGCATTACTATTAGTGTC
1, 2


 


156
HPV16-2082-35
TTGCACAATCCTTTACAATTTTTGCCTGTGAATTA
1, 2


 


157
HPV16-2117-35
TTTTTTTTCTGCTCGTTTATAATGTCTACACATTG
1, 2


 


158
HPV16-2152-35
CATCTATATTTTATCCATTGACTCATACTCATTTG
1, 2


 


159
HPV16-2187-35
TTTGCTTCCAATCACCTCCATCATCTACCCTATCA
1, 2


 


160
HPV16-2222-35
AAACTCTACACCTTGATACCTTAAAAACATAACAA
1, 2


 


161
HPV16-2257-35
TGCAAAAATCTTTTTAATGCAGTTAAAAATGACAT
1, 2


 


162
HPV16-2292-35
CATATAGTAATATGCAATTTTTTTTAGGTATGCCT
1, 2


 


163
HPV16-2327-35
CATACCAAATAATGATTTACCTGTGTTAGCTGCAC
1, 2


 


164
HPV16-2362-35
CATATTACAGACCCTTGCAGAAATTTCATTAAACT
1, 2


 


165
HPV16-2397-35
GTTGTAACCAAAAATGGCTTTTAGAATTTACAAAA
1, 2


 


166
HPV16-2432-35
ATCATCTAACATACCTATTTTGGCATCTGCTAATG
1, 2


 


167
HPV16-2467-35
TTGTCATCTATGTAGTTCCAACAGGGCACTGTAGC
1, 2


 


168
HPV16-2502-35
TAGAAACTAAATTTCCATCCAATGCATTTCTTAAA
1, 2


 


169
HPV16-2537-35
TTTTAGTTGTACCAATGGTCTATGCTTTACATCCA
1, 2


 


170
HPV16-2572-35
GCATTAATGTTAGATGTAATTAATAATGGAGGGCA
1, 2


 


171
HPV16-2607-35
TATTATGTAAATAAGGCCACCTAGAATCTGTACCA
1, 2


 


172
HPV16-2642-35
TGGAAACTCATTAGGAAATGTAAACACCACCAATC
1, 2


 


173
HPV16-2677-35
TTAAGCTCATACACTGGATTTCCGTTTTCGTCAAA
1, 2


 


174
HPV16-2709-32
TCCTTGAGAAAAAGGATTTCCAGTTCTTATCA
1, 2


 


175
HPV16-2743-34
TCCTCGTCCTCGTGCAAACTTAATCTGGACCACG
1


 


176
HPV16-2777-34
AAACGTTGGCAAAGAGTCTCCATCGTTTTCCTTG
1


 


177
HPV16-2810-33
TAATGTGTTAGTATTTTGTCCTGACACACATTT
1


 


178
HPV16-2844-34
TATATGGTCACGTAGGTCTGTACTATCATTTTCA
1


 


179
HPV16-2878-34
TAGCACATTCTAGGCGCATGTGTTTCCAATAGTC
1


 


180
HPV16-2912-34
TGTTTAAATCCCATTTCTCTGGCCTTGTAATAAA
1


 


181
HPV16-2945-33
ACAGCCAGTGTTGGCACCACTTGGTGGTTAATA
1


 


182
HPV16-2979-34
CAGTTCAATTGCTTGTAATGCTTTATTCTTTGAT
1


 


183
HPV16-3013-34
ATTGTGAGTTATATATTGTTTCTAACGTTAGTTG
1


 


184
HPV16-3088-40
TTTTTATACATCCTGTTGGTGCAGTTAAATACACTTCAAG
1


 


185
HPV16-3129-41
TATGTCTCCATCAAACTGCACTTCCACTGTATATCCATGTT
1


 


186
HPV16-3169-40
TATATATATGTGTCCAGTTTGTATAATGCATTGTATTGCA
1


 


187
HPV16-3204-35
ACCCTCTACCACAGTTACTGATGCTTCTTCACAAA
1


 


188
HPV16-3235-31
ATGAACATAATATAAACCATAATAGTCAACTTG
1


 


189
HPV16-3301-29
CTTTATTTTTACTATATTTTTCTGCATCA
1


 


190
HPV16-3329-28
ACCTGACCACCCGCATGAACTTCCCATA
1


 


191
HPV16-3358-29
TGCTAAACACAGATGTAGGACATAATATT
1


 


192
HPV16-3425-32
GTATGGGTCGCGGCGGGGTGGTTGGCCAAGTG
1


 


193
HPV16-3460-35
TCTGTGTTTCTTCGGTGCCCAAGGCGACGGCTTTG
1


 


194
HPV16-3494-34
GTGTCTGGCTCTGATCTTGGTCGCTGGATAGTCG
1


 


195
HPV16-3528-34
GTGCAACAACTTAGTGGTGTGGCAGGGGTTTCCG
1


 


196
HPV16-3562-34
CAGTGAGGATTGGAGCACTGTCCACTGAGTCTC
1


 


197
HPV16-3598-36
TACAGTTAATCCGTCCTTTGTGTGAGCTGTTAAATG
1


 


198
HPV16-3632-34
CCTTTTAAATGTACTATGGGTGTAGTGTTACTAT
1


 


199
HPV16-3667-35
ATCTATATCTTAAACATTTTAAAGTATTAGCATCA
1


 


200
HPV16-3703-36
ACGACACTGCAGTATACAATGTACAATGCTTTTTAA
1


 


201
HPV16-3738-35
ATGTTTTACATTATGTCCTGTCCAATGCCATGTAG
1


 


202
HPV16-3774-36
TTCACTATCATATGTAAGTGTAACAATTGCACTTTT
1


 


203
HPV16-3812-38
GGTATTTTAACTTGAGACAAAAATTGGTCACGTTGCCA
1


 


204
HPV16-3850-38
ATATAGACATAAATCCAGTAGACACTGTAATAGTTTTT
1


 


205
HPV16-3918-33
CACAAAAGCACACAAAGCAAAGCAAAAAGCACG
1


 


206
HPV16-3952-34
AGACAAAAGCAGCGGACGTATTAATAGGCAGACA
1


 


207
HPV16-3988-36
TAATACCAATATTATTAATGATGTGTATGTAGACAC
1


 


208
HPV16-4023-35
CACCTAAACGCAGAGGCTGCTGTTATCCACAATAG
1


 


209
HPV16-4059-36
AATGGTATATAAACAAATATAATATATACAATAAAA
1


 


210
HPV16-4094-35
TAATTAAAAAGCGTGCATGTGTATGTATTAAAAAT
1


 


211
HPV16-4130-36
ATATGTAACAATTACATTATGTACATATACATTATG
1


 


212
HPV16-4165-35
AAAGAAAAAATAGTAAGTTATGGTATACAACAATT
1


 


213
HPV16-4201-36
AAACAAACAAAAAAAAAAATTATATATGAAAATAAA
1


 


214
HPV16-4236-35
ATTGTTAAGTAATAACAGTTTATTAAAAAACAAAC
1, 2


 


215
HPV16-4271-35
CACGTTTTGTGCGTTTTGCAGAACGTTTGTGTCGC
1, 2


 


216
HPV16-4306-35
CTGTTTGCATGTTTTATAAAGTTGGGTAGCCGATG
1, 2


 


217
HPV16-4341-35
ACCTTAGGTATAATGTCAGGTGGACATGTACCTGC
1, 2


 


218
HPV16-4376-35
ATTGTAATATTTGTTCAGCAATAGTTTTGCCTTCA
1, 2


 


219
HPV16-4411-35
TCCTAACCCACCAAAAAATACACCCATACTTCCAT
1, 2


 


220
HPV16-4446-35
CCAGTGCGTCCGCCTGTACCCGACCCTGTTCCAAT
1, 2


 


221
HPV16-4481-35
TAGCTGTGGGAGGCCTTGTTCCCAATGGAATATAC
1, 2


 


222
HPV16-4516-35
TGTTAAAGGGGGTCTTACAGGAGCAAGTGTATCTG
1, 2


 


223
HPV16-4551-35
ACTATAGAAGGATCAGAAGGGCCCACAGGATCTAC
1, 2


 


224
HPV16-4586-35
CAGCATCAATAAAACTAGTTTCTTCCACTAAAGAA
1, 2


 


225
HPV16-4621-35
ATCTGGGGGAATGGAAGGTACAGATGTTGGTGCAC
1, 2


 


226
HPV16-4656-35
GTATCAGTTGAAGTAGTAATACTAAATCCTGATAC
1, 2


 


227
HPV16-4691-35
TAACAGTATTATTAATATCTAATATAGCAGGTGTG
1, 2


 


228
HPV16-4726-35
AGTGAAAGTGGGATTATTATGTGTAGTAACAGTAG
1, 2


 


229
HPV16-4762-36
TTCTGCAGGTGTTGGAGGCTGCAATACAGATGGGTC
1, 2


 


230
HPV16-4798-36
AATAGTGGATGATGAAAGTGTAAAATGCCCTCCAGT
1, 2


 


231
HPV16-4834-36
TGTATCCATAGGAATTTCTTCATAATTATGTGTACT
1, 2


 


232
HPV16-4870-36
ACTAGTTACTGTGTTAGGGTTTGTGCTAACAATAAA
1, 2


 


233
HPV16-4906-36
GCGTGCCACTGGGCGAGACCCTGGTATGGGTGTGCT
1, 2


 


234
HPV16-4975-34
AGTGGGAGTGGTTACAAAAGCAGGGTCTACAACT
1, 2


 


235
HPV16-5010-35
CCTTCATATGCAGGATTATCATATGTAATAAGTTT
1, 2


 


236
HPV16-5045-35
TACTAGAAAAATATAATGTATTATCCACATCTATA
1, 2


 


237
HPV16-5080-35
GTCAGGATCTGGAGCTATATTAATACTATTATCAT
1, 2


 


238
HPV16-5115-35
AATGCTGGCCTATGTAAAGCAACTATATCCAAAAA
1, 2


 


239
HPV16-5150-35
TTCTACTGTACCTAATGCCAGTACGCCTAGAGGTT
1, 2


 


240
HPV16-5185-35
TCCACTACGAGTACGTAGTGTTTGTTTATTACCAA
1, 2


 


241
HPV16-5220-35
TCATAATAATAATGTACCTTAGCACCTATAGATTT
1, 2


 


242
HPV16-5255-35
ATTCTATTTCTTCTGCAGGATCAATAGTACTTAAA
1, 2


 


243
HPV16-5290-35
AGTGGTAGTATATGTAGAAGGTGTTATAGTTTGTA
1, 2


 


244
HPV16-5325-35
CCATTATTAATAGAAGTAGGTGAGGCTGCATGTGA
1, 2


 


245
HPV16-5360-35
CTGTAATAAAGTCATCTGCATAAATATCATATAAT
1, 2


 


246
HPV16-5395-35
AGAGGGTACAGATGGTACCGGGGTTGTAGAAGTAT
1, 2


 


247
HPV16-5430-35
GTTGTATTTGCAGGAATATAACCTGATAAAGATGT
1, 2


 


248
HPV16-5465-35
CTAAAGGAATATTGTATGCACCACCAAAAGGAATT
1, 2


 


249
HPV16-5500-35
GTCAGTTATATTAATGGGTATATCAGGACCTGATA
1, 2


 


250
HPV16-5535-35
GACCCTGGAACTATAGGAATTAATGAAGGAGCTTG
1, 2


 


251
HPV16-5570-35
AGTCACCTGCATCAGCAATAATTGTATATTGTGGA
1, 2


 


252
HPV16-5604-34
TTTCGTAACATGTAATAACTAGGATGTAAATAAA
1, 2


 


253
HPV16-5638-34
ATCTGAAAAAAAATATGGTAAACGTTTACGTCGT
1, 2


 


254
HPV16-5675-37
CAAGTAGACAGTGGCCTCACTAGGCAGCCAAAGAGAC
1, 2


 


255
HPV16-5713-38
TCATCCGTGCTTACAACCTTAGATACTGGGACAGGAGG
1, 2


 


256
HPV16-5750-37
TCCTGCATGATAATATATGTTTGTGCGTGCAACATAT
1, 2


 


257
HPV16-5788-38
GGAAAATAGGGATGTCCAACTGCAAGTAGTCTGGATGT
1, 2


 


258
HPV16-5823-35
GAACTAATATTTTGTTATTGTTAGGTTTTTTAATA
1, 2


 


259
HPV16-5857-34
CTAAATACCCTGTATTGTAATCCTGATACTTTAG
1, 2


 


260
HPV16-5891-34
AAAACCAAACTTATTGGGGTCAGGTAAATGTATT
1, 2


 


261
HPV16-5926-35
CGCTGTGTATCTGGATTATAAAATGAGGTGTCAGG
1, 2


 


262
HPV16-5960-34
ACCTACCTCAACACCTACACAGGCCCAAACCAGC
1, 2


 


263
HPV16-5994-34
GGCCACTAATGCCCACACCTAATGGCTGACCACG
1, 2


 


264
HPV16-6029-35
ATTTTCTGTGTCATCCAATTTATTTAATAAAGGAT
1, 2


 


265
HPV16-6063-34
TATCCACACCTGCATTTGCTGCATAAGCACTAGC
1, 2


 


266
HPV16-6097-34
GTTTGTTTGTAATCCATAGATATACATTCTCTAT
1, 2


 


267
HPV16-6132-35
CTATAGGTGGTTTGCAACCAATTAAACACAATTGT
1, 2


 


268
HPV16-6166-34
TTGGTACATGGGGATCCTTTGCCCCAGTGTTCCC
1, 2


 


269
HPV16-6200-34
TAATGGTGGACAATCACCTGGATTTACTGCAACA
1, 2


 


270
HPV16-6235-35
ATATCACCATCCTGAATAACTGTGTTTATTAACTC
1, 2


 


271
HPV16-6269-34
AGTAAAGTCCATAGCACCAAAGCCAGTATGAACC
1, 2


 


272
HPV16-6303-34
CCAGTGGAACTTCACTTTTGTTAGCCTGTAATGT
1, 2


 


273
HPV16-6338-35
ATAATCTGGATATTTGCAAATAGATGTACAAATAT
1, 2


 


274
HPV16-6372-34
AGCTGTCGCCATATGGTTCTGACACCATTTTAAT
1, 2


 


275
HPV16-6406-34
ACAAACATTTGTTCCCTTCGTAAATAAAAAAATA
1, 2


 


276
HPV16-6441-35
CACCAACAGTACCAGCCCTATTAAATAAATGTCTA
1, 2


 


277
HPV16-6475-34
GAGCCTTTAATGTATAAATCGTCTGGTACATTTT
1, 2


 


278
HPV16-6546-36
CATCAGAGGTAACCATAGAACCACTAGGTGTAGGAA
1, 2


 


279
HPV16-6582-36
CTCGTTGTAACCAATAAGGTTTATTGAATATTTGGG
1, 2


 


280
HPV16-6618-36
GGTTACCCCAACAAATGCCATTATTGTGGCCCTGTG
1, 2


 


281
HPV16-6653-35
ACTGCGTGTAGTATCAACAACAGTAACAAATAGTT
1, 2


 


282
HPV16-6689-36
TGAAGTAGATATGGCAGCACATAATGACATATTTGT
1, 2


 


283
HPV16-6725-36
GTACTCCTTAAAGTTAGTATTTTTATATGTAGTTTC
1, 2


 


284
HPV16-6761-36
AATAAACTGTAAATCATATTCCTCCCCATGTCGTAG
1, 2


 


285
HPV16-6796-35
ACGTCTGCAGTTAAGGTTATTTTGCACAGTTGAAA
1, 2


 


286
HPV16-6832-36
AAAATAGTGGAATTCATAGAATGTATGTATGTCATA
1, 2


 


287
HPV16-6868-36
CCTGGGGGAGGTTGTAGACCAAAATTCCAGTCCTCC
1, 2


 


288
HPV16-6903-35
GGGTTACAAACCTATAAGTATCTTCTAGTGTGCCT
1, 2


 


289
HPV16-6939-36
GTGCTGGAGGTGTATGTTTTTGACAAGCAATTGCCT
1, 2


 


290
HPV16-6975-36
AAAAAGTGTATTTTTTAAGGGGATCATCTTCTTTAG
1, 2


 


291
HPV16-7011-36
GGTCTGCAGAAAACTTTTCCTTTAAATTTACTTCCC
1, 2


 


292
HPV16-7047-36
GTAGTAAAAATTTGCGTCCTAAAGGAAACTGATCT
1, 2


 


293
HPV16-7082-35
TAATGTAAATTTTGGTTTGGCCTTCAATCCTGCTT
1, 2


 


294
HPV16-7115-33
AGATGAGGTGGTGGGTGTAGCTTTTCGTTTTCC
1, 2


 


295
HPV16-7141-25
CACATACAACTTAATATTTACAAGC
1, 2


 


296
HPV16-7148-33
CTTACGTTTTTTGCGTTTAGCAGTTGTAGAGGT
1, 2


 


297
HPV16-7181-33
AACACTAATTCAACATACATACAATACTTACAG
1, 2


 


298
HPV16-7216-35
ACATACAAGCACATACAAACATATACACAACAAAC
1, 2


 


299
HPV16-7265-24
TTTATTATACCATACATACAAACA
1, 2


















TABLE 7





SEQ




ID




NO
Name
Sequence







300
HPV16 CP1
ACAACTATACATGATATAATATTAGAATGTGTGTAC


 


301
HPV16 CP2
TCTACTGTTATGAGCAATTAAATGACAGCTCAGAGG


 


302
HPV16 CP3
TGGAAGACCTGTTAATGGGCACACTAGGAATTGTGT


 


303
HPV16 CP4
GTATATCAAATATTAGTGAAGTGTATGGAGACACGC


 


304
HPV16 CP5
CCTTGAAGTGTATTTAACTGCACCAACAGGATGTAT


 


305
HPV16 CP6
ACTTGGCCAACCACCCCGCCGCGACCCATACCAAAG


 


306
HPV16 CP7
GCGTGCTTTTTGCTTTGCTTTGTGTGCTTTTGTGTG


 


307
HPV16 CP8
AAGTTGTAGACCCTGCTTTTGTAACCACTCCCACTA


 


308
HPV16 CP9
TTCCTACACCTAGTGGTTCTATGGTTACCTCTGATG









Bead/Probe Conjugation
Magnetic streptavidin beads (5% solids, Seradyn, Inc., Indianapolis, Ind.), were vortexed and washed twice in a Tween-based wash buffer. They were then incubated with a cocktail of biotinylated capture probes comprising 180 nM/probe and incubated at 37° C. for 30 min with shaking at 1150 RPM. The beads were pelleted, washed three more times, and resuspended in a casein blocking solution for storage at 4° C.
E2 Integrity Assay
20 μl of Qiazol extracted RNA in 30 μl of a chaotropic salt solution was captured onto 10 μl of the bead-conjugated capture probes for 30 min at 60° C. with shaking. After this step the sample was split by pipetting 30 μl of the reaction into two separate clean wells on the same 96-well plate. A magnetic rack was then used to pull the bead-probe-RNA complex down and the resulting pellet was washed with a buffered saline-detergent solution twice, once for 2 min and once for 5 min. The signal probe cocktails, one with all probes and one without E2-region probes, were then added at 4.2 nM in 65 μl of a nucleic acid hybridization buffer and the hybridization reaction was performed for 30 min at 60° C. with shaking. After this reaction the bead complex was again pelleted and the plate was dried on absorbent paper towels. A solution of anti-DNA:RNA nucleic acid hybrid antibody conjugated with alkaline phosphatase, was added at 45 μl/well and the plate was incubated for 30 min at 45°. The beads were again pelleted and washed five times for one min/wash, this time with a Tris-based wash buffer. 35 μl of chemiluminescent alkaline phosphatase substrate was then added and the plate was shaken at 350 RPM for 15 min at room temperature under a foil seal to protect the samples from light. The luminescence from the wells was then read on a QIAGEN® DML 3000TM microplate luminometer (Qiagen Gaithersburg, Inc., Gaithersburg, Md.).
The signal:noise (“S/N”) of the sample values in the two wells (with and without E2 probes) was determined. Samples with a signal:noise value below 2 in the all-probes well were excluded from statistical analyses. The extent of E2 disruption was determined with the following formula percentage difference: [(S/N(all)−S/N(no E2))/S/N(all)]×100.
Example 19
An in vitro transcribed HPV E6/E7 was provided as described above. Three biotinylated capture probes of 40 nucleotides each, evenly spaced along the transcript were conjugated to magnetic streptavidin beads as described above. 1×105 copies of the HPV 16 E6/E7 transcript was then captured to the streptavidin beads as described above. Probe cocktails comprising 0, 5, 10, 15, and 19 signal probes for HPV E6/E7 were generated. Excluding the capture probe, 19 signal probes is sufficient to hybridize to the full length of the E6/E7 transcript. Additionally, a probe cocktail comprising 19 signal probes for HPV E6/E7 plus 5 signal probes for HPV 16 L1 was generated. The S/N ratio was calculated for each probe cocktail. Results are shown at FIG. 13. As can be seen, signal intensity increases in a somewhat linear fashion with increasing numbers of probes.
Example 20
E2 integrity in SiHa and W12 cells was compared using the methods described at Example 18. SiHa cells comprise an integrated HPV 16 genome, resulting in disruption of the majority of the E2 gene maintained in the cell. In contrast, the HPV 16 genome is maintained in episomal form in W12 cells; thus the E2 gene is intact. Data are shown at FIG. 22. When E2 probes are removed from the signal amplification cocktail, there is no drop in signal in SiHa cells because none of the signal with all of the probes is from the E2 region. However, the signal from W12 drops significantly, indicating that approximately 50% of the RNA transcripts detected are from E2.
Example 21
LSIL and HSIL samples were tested in the E2 integrity assay described at Example 18. Data are shown at FIG. 23 and summarized below at Table 8.














TABLE 8







Level
Minimum
25%
Median
75%
Maximum























HSIL
2
11
22.9
38.1
50.2



LSIL
16.5
34
42.9
47.7
57.2










As can be seen, there is a significant difference between LSIL and HSIL samples (p=0.0012). More noteworthy, however, is the distribution of the samples in each lesion category. While the maximum percentage difference for the lesion categories is similar, the minimums are much more different (2 for HSIL and 16.5 for LSIL). This pattern makes sense given that only a small percentage of HSIL samples eventually progress to cervical cancer.

## Claims

1. A method of detecting the presence of a target HPV RNA, the method comprising:
a) providing at least one DNA capture probe;
b) providing a first anti-RNA:DNA hybrid antibody, wherein the first anti-RNA:DNA hybrid antibody is bound to a support;
c) hybridizing the target HPV RNA to said at least one DNA capture probe, yielding a target RNA:DNA capture probe complex;
d) incubating said target HPV RNA:DNA capture probe complex with said anti-RNA:DNA hybrid antibody, yielding a bound target HPV RNA:DNA capture probe complex;
e) providing at least one DNA amplification probe, and hybridizing said at least one DNA amplification probe to said bound target HPV RNA:DNA capture probe complex, yielding a bound target HPV RNA:DNA capture/amplification probe complex;
f) providing a second anti-RNA:DNA hybrid antibody, and incubating said bound target HPV RNA:DNA capture/amplification probe complex with said second anti-RNA:DNA hybrid antibody, yielding a bound target HPV RNA:DNA:antibody complex;
g) detecting said second anti-RNA:DNA hybrid antibody, wherein said detecting indicates the presence of said target HPV RNA,

wherein at least one of the capture probes comprises an isolated nucleic acid having an overall length of not more than 50 nucleotides, wherein the isolated nucleic acid comprises 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: 20 and SEQ ID NO: 300 to SEQ ID NO: 308, RNA equivalents thereof, and full complements thereof;
wherein the capture probe is not capable of hybridizing to more than one type of HPV genome;
wherein the amplification probe comprises at least one nucleotide sequence having at least 75-percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 105 and SEQ ID NO: 111 to SEQ ID NO: 299; and
wherein the target HPV RNA is not amplified.

2. The method of claim 1, wherein the target HPV RNA is a splice variant, and wherein the at least one DNA capture probe and the at least one DNA amplification probe are selected to detect the presence of said splice variant.

3. A method for determining whether a target nucleic acid is absent from or disrupted in a sample, said method comprising:
a) treating a first portion of the sample under conditions sufficient to induce the formation of:
i) a first set of DNA:RNA hybrids comprising the target nucleic acid; and
ii) a second set of DNA:RNA hybrids comprising a reference nucleic acid;

b) treating a second portion of the sample under conditions sufficient to induce the formation of the second set of DNA:RNA hybrids, but not the first set of DNA:RNA hybrids;
c) generating a detectable signal in the first portion of the sample and the second portion of the sample, wherein the detectable signal has an intensity that correlates with the concentration of DNA:RNA hybrids; and
d) comparing the intensity of the detectable signal in the first portion of the sample and the intensity of the detectable signal in the second portion of the sample, wherein:
i) the target nucleic acid is intact and present in the sample if the intensity of the detectable signal in the first portion of the sample is greater than the intensity of the detectable signal in the second portion of the sample; and
ii) the target nucleic acid is absent from the sample if the intensity of the detectable signal in the first portion of the sample is less than or equal to the intensity of the detectable signal in the second portion of the sample;


wherein the first portion of the sample and the second portion of the sample are formed by a method comprising contacting the sample with a first capture probe set specific for the target nucleic acid, wherein hybridization of the first capture probe set to the target nucleic acid generates a first capture complex; and a second capture probe set specific for the reference nucleic acid, wherein hybridization of the second capture probe set to the reference nucleic acid generates a second capture complex;
wherein the first capture probe set comprises at least two capture probes having an overall length of not more than 50 nucleotides, wherein each capture probe comprises at least one nucleotide sequence having at least 75-percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56 to SEQ ID NO: 59, and SEQ ID NO: 111 to SEQ ID NO: 299, RNA equivalents thereof, and full complements thereof;
wherein the second capture probe set comprises at least two capture probes having an overall length of not more than 50 nucleotides, wherein each capture probe comprises at least one nucleotide sequence having at least 75-percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56 to SEQ ID NO: 59, SEQ ID NO: 111 to SEQ ID NO: 174, SEQ ID NO: 214 to SEQ ID NO: 299, RNA equivalents thereof, and full complements thereof;
wherein the first capture probe comprises: i) a region capable of hybridizing to the target nucleic acid; and ii) a region capable of hybridizing to a first nucleic acid sequence of an anchor probe; and the second capture probe comprises: i) a region capable of hybridizing to the target nucleic acid; and ii) a region capable of hybridizing to a second nucleic acid sequence of an anchor probe, and
wherein the anchor probe is bound to or adapted to be bound to a first support and/or second support.

4. The method of claim 3, further comprising capturing the first capture complex and the second capture complex to a support.

5. The method of claim 4, wherein the first and second capture probes are bound to or adapted to be bound to the support.

6. The method of claim 5, wherein the capture probes comprise a ligand and the support comprises a ligand-binding moiety.

7. The method of claim 6, wherein the first capture probe and the second capture probe comprise the same ligand.

8. The method of claim 6, wherein the first capture probe and the second capture probe comprise different ligands, wherein:
a) the first portion of the sample is contacted with a first set of solid supports comprising a ligand binding moiety capable of binding the ligand of the first capture probe and a ligand binding moiety capable of binding the ligand of the second capture probe; and
b) the second portion of the sample is contacted with a second set of solid supports comprising a ligand binding moiety capable of binding the ligand of the second capture probe, but not the first capture probe.

9. The method of claim 8, wherein the first portion of the sample and the second portion of the sample is contacted with
i) a first probe set comprising a plurality of signal probes capable of hybridizing to the target nucleic acid; and
ii) a second probe set comprising a plurality of signal probes capable of hybridizing to the reference nucleic acid.

10. The method of claim 6, wherein the first capture probe and the second capture probe are biotinylated and wherein the first support and the second support comprise a biotin-binding moiety.

11. The method of claim 6, wherein the capture probes are covalently bound to the support.

12. The method of claim 4, wherein:
a) the first and second capture complexes comprise a DNA:RNA hybrid; and
b) the first and second capture complexes are captured to the first and second supports by a method comprising contacting the first and second capture complexes with an entity capable of specifically binding to a DNA:RNA hybrid, wherein the entity capable of specifically binding to a DNA:RNA hybrid is bound to the support or adapted to be bound to the support.

13. The method of claim 12, wherein the entity capable of specifically binding to a DNA:RNA hybrid comprises a ligand and the first and second supports comprise a ligand-binding moiety.

14. The method of claim 13, wherein the entity capable of specifically binding to a DNA:RNA hybrid is biotinylated and wherein the support comprises a biotin-binding moiety.

15. The method of claim 12, wherein the entity capable of specifically binding to a DNA:RNA hybrid is covalently bound to the support.

16. The method of claim 12, wherein the entity capable of specifically binding to a DNA:RNA hybrid is a DNA:RNA hybrid-specific antibody or a fragment thereof.

17. The method of claim 3, wherein the first nucleic acid sequence and the second nucleic acid sequence are the same.

18. The method of claim 3, wherein the first nucleic acid sequence and the second nucleic acid sequence are different.

19. The method of claim 18, wherein the first nucleic acid sequence and the second nucleic acid sequence are disposed in the same anchor probe.

20. The method of claim 18, wherein the first nucleic acid sequence and the second nucleic acid sequence are disposed in different anchor probes.

21. The method of claim 3, wherein:
a) the first support comprises an anchor probe comprising the first nucleic acid sequence and an anchor probe comprising second nucleic acid sequence; and
b) the second support comprises anchor probes comprising the second nucleic acid sequence, but does not comprise anchor probes comprising the first nucleic acid sequence.

22. The method of claim 3, wherein:
a) the first set of DNA:RNA hybrids is formed by a method comprising contacting the sample with a first signal probe capable of hybridizing to the target nucleic acid; and
b) the second set of DNA:RNA hybrids is formed by a method comprising contacting the sample with a second signal probe capable of hybridizing to the reference nucleic acid.

23. The method of claim 22, wherein:
a) the first portion of the sample is contacted with the first signal probe and the second signal probe; and
b) the second portion of the sample is contacted with the second signal probe, but not the first signal probe,

wherein the first signal probe is specific for the target nucleic acid and the second signal probe is specific for the reference nucleic acid.

24. The method of claim 13, wherein:
a) the first signal probe is disposed in a first probe set comprising a plurality of signal probes capable of hybridizing to the target nucleic acid; and
b) the second signal probe is disposed in a second probe set comprising a plurality of signal probes capable of hybridizing to the reference nucleic acid.

25. The method of claim 24, wherein:
a) the plurality of signal probes of the first probe set is capable of hybridizing to at least 70% of the target nucleic acid;
b) the plurality of signal probes of the second probe set is capable of hybridizing to at least 70% of the reference nucleic acid.

26. The method of claim 3, wherein the detectable signal is generated by a method comprising contacting the first portion of the sample and the second portion of the sample with an entity capable of specifically binding to a DNA:RNA hybrid.

27. The method of claim 26, wherein the entity capable of specifically binding a DNA:RNA hybrid is an DNA:RNA hybrid-specific antibody or a fragment thereof.

28. The method of claim 3, further comprising:
a) generating the first portion of the sample and the second portion of the sample by a method comprising:
i) contacting the sample with at least a first biotinylated capture probe specific for the target nucleic acid;
ii) contacting the sample with at least a second biotinylated capture probe specific for the reference nucleic acid;
iii) contacting the sample with a streptavidin-coated magnetic bead under conditions sufficient to permit binding of the biotinylated capture probes to the streptavidin coated bead; and
iv) separating the streptavidin coated beads into separate containers to form the first portion of the sample and the second portion of the sample;

b) forming the first set of DNA:RNA hybrids and the second set of DNA:RNA hybrids in the first portion of the sample by a method comprising contacting the first portion of the sample with a probe cocktail comprising:
i) a plurality of detectably labeled nucleic acid probes capable of hybridizing to the target nucleic acid, wherein said plurality is sufficient to cover the target nucleic acid; and
ii) a plurality of detectably labeled nucleic acid probes capable of hybridizing to the reference nucleic acid, wherein said plurality is sufficient to cover the target nucleic acid; and

c) forming the second set of DNA:RNA hybrids in the second portion of the sample by a method comprising contacting the second portion of the sample with a probe cocktail comprising a plurality of detectably labeled signal probes capable of hybridizing to the reference nucleic acid, wherein said plurality is sufficient to cover the target nucleic acid,

wherein the detectable signal is generated by the detectably labeled signal probes.

29. The method of claim 3, wherein:
a) the target nucleic acid is an HPV E2 nucleic acid; and
b) the reference nucleic acid is selected from the group consisting of:
i) HPV E1 nucleic acid
ii) HPV E6/E7 nucleic acid
iii) HPV L1 nucleic acid
iv) HPV L2 nucleic acid.

30. The method of claim 29, wherein a group of reference nucleic acids are detected, the group comprising at least two reference nucleic acids selected from the group consisting of:
i) HPV E1 mRNA or cDNA
ii) HPV E6/E7 mRNA or cDNA
iii) HPV L1 mRNA or cDNA; and
iv) HPV L2 mRNA or cDNA.

31. The method of claim 30, wherein the group of reference nucleic acids comprises:
i) HPV E1 mRNA;
ii) HPV E6/E7 mRNA;
iii) HPV L1 mRNA; and
iv) HPV L2 mRNA.

32. A method of predicting the onset of HPV-induced cell transformation in a patient, said method comprising detecting the presence or absence of an HPV E2 mRNA in an HPV-infected tissue derived from the patient by the method of claim 29, wherein the absence of HPV E2 mRNA is indicative of the onset of HPV-induced cell transformation.

33. A method of detecting integration of an HPV genome into a genome of a host cell, said method comprising detecting the presence or absence of an HPV E2 mRNA in an HPV-infected tissue derived from the patient by the method of claim 29, wherein the absence of HPV E2 mRNA is indicative of integration of the HPV genome into a genome of a host cell.