WO2005100610A2 - Virus detection method, primers therefor and screening kit - Google Patents

Virus detection method, primers therefor and screening kit Download PDF

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Publication number
WO2005100610A2
WO2005100610A2 PCT/GB2005/001366 GB2005001366W WO2005100610A2 WO 2005100610 A2 WO2005100610 A2 WO 2005100610A2 GB 2005001366 W GB2005001366 W GB 2005001366W WO 2005100610 A2 WO2005100610 A2 WO 2005100610A2
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virus
primer
sequence
self
dna
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WO2005100610A3 (en
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Keith Hart
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University College Cardiff Consultants Ltd
Cardiff University
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University College Cardiff Consultants Ltd
Cardiff University
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Priority claimed from GB0410892A external-priority patent/GB0410892D0/en
Priority claimed from GB0423138A external-priority patent/GB0423138D0/en
Priority claimed from GB0425085A external-priority patent/GB0425085D0/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

  • the present invention relates to an improved method for detecting, typing and characterising the presence of viruses, particularly those that have been associated with carcinogenic activity in mammals, such as human papillomavirus, particularly, but not exclusively, in cervical samples and to primers and probes for use in the method.
  • the invention further relates to a diagnostic kit and screening method, which uses the kit.
  • the infecting virus type per cell ie the number of viral copies per cell or ratio of viral genome : human genome
  • the integration state ie whether the viral DNA is 'free' in the cytosol of the cell or integrated into the host genome
  • Cervical cancer is the second most frequent cause of death from cancer in women, worldwide. Cervical screening programmes reduce the incidence of cervical cancer; however, 50% of invasive cervical cancers arise in women screened using existing cytological methodologies.
  • HPV human papillomaviruses
  • HPV detection and typing techniques have been proposed as an adjunct to, or replacement for, the current cytological screening regime.
  • the success of such strategies will depend on the development of rapid, reliable, sensitive and specific HPV-detection methods applicable in the clinical setting.
  • HPV is genetically composed of early (E) and late (L) genes, which are functionally divided into several open reading frames (ORFs): viral replication (E1), regulation of transcription (E2), coding for cytoplasmic proteins (E4) and malignant transformation (E5, E6, E7) as early genes, and coding for capsid proteins (L1 , L2) as late genes.
  • ORFs open reading frames
  • the general primers GP5 ⁇ '-TTT GTT ACT GTG GTA GAT AC-3'
  • GP6 (3'-ACT AAA TGT CAA ATA AAA AG-5') (Snijders et al, J.Gen. Virol.
  • a colorimetric microtitre plate based hybridisation assay was developed.
  • the method used one biotinylated primer (bio-GP6+) in the GP-PCR.
  • Biotinylated PCR products were captured on streptavidin-coated microtitre plates, denaturated and hybridised to digoxigenin-(DIG-) labelled HPV-specific internal oligonucleotide probes.
  • the DIG-labelled hybrids were detected using an enzyme immunoassay (EIA).
  • EIA enzyme immunoassay
  • PCR-EIA 6 low-risk HPVs following a general primer GP5+/bioGP6+-mediated PCR. This technique is hereinafter referred to as PCR-EIA.
  • HR/LR high-risk/low risk
  • Some fluorogenic probe assays for HPV DNAs are based on the PCR amplification of a portion of the L1 open reading frames of HPV-16, -18, -31 , -33, and -35 DNAs by using genotype-specific probes that bind to the amplified DNA.
  • the probes are blocked at their 3' termini and hence cannot be extended by the polymerase. If, during the course of primer extension, Taq polymerase encounters a bound probe, its 5'- 3' exonuclease activity degrades the probe, releasing the 5' fluor from the 3' quencher. This causes an increase in the fluorescence emitted by the reporter which, in the presence of an excess amount of the probe, is directly related to the amount of
  • HPV DNA present in the sample before amplification.
  • 'Molecular beacons' comprise a probe flanked by a hairpin loop that holds a fluorophore and quencher in close proximity until specific binding of the probe to its target opens out the structure, producing a fluorescent signal.
  • HPV DNA is present as an episomal form in low grade cervical intra- epithelial neoplasia (CIN) lesions, and the principal form of the viral DNA in high grade CIN and invasive cancers is integrated into the host genome. In most cervical carcinomas, HPV genomes are integrated into host cell chromosomes and transcribed into mRNAs encompassing viral and cellular sequences.
  • CIN cervical intra- epithelial neoplasia
  • HPV genomes persist as episomes, and derived transcripts contain exclusively viral sequences.
  • detection of HPV transcripts derived from integrated HPV genomes may specifically indicate both CIN lesions at high-risk for progression as well as invasive cervical cancers.
  • E6 and E7 proteins can activate some oncogenes and/or inactivate host tumour suppressor gene products (p53, pRB), resulting in uncontrolled cellular growth and malignant transformation.
  • Integration of HPV DNA is therefore considered an important genetic change in the process of cervical carcinogenesis, and being able to determine the level of integration is believed to be important, diagnostically.
  • Some detection techniques therefore focus on identifying and quantifying disruptions in the early genes, particularly E1/E2 and E6/E7. These have been carried out using techniques such as multiplex PCR (where primers for each sequence are included in the same reaction) and RT-PCR that allows discrimination of HPV mRNAs derived from integrated and episomal viral genomes.
  • Hitherto-known techniques therefore require a plurality of techniques and/or probes to detect HPV types and integration status, which is time-consuming, slow and generally unsatisfactory for clinical use. Furthermore, none of these techniques satisfactorily deal with the determination of viral load, since none result in a measure of the number of viral copies per cell. These systems have used different methods for the quantitation of virus copy number and cell copy number and thus the two are not strictly comparable. Previous methods incorporating assays for the ⁇ -globin or other housekeeping genes of the genomic DNA have, at most, given rise to a range of viral DNA copies per microgram of cellular DNA, rather than per cell. Other uses of housekeeping genes have been as PCR controls or to determine PCR limits and not to provide information about viral load.
  • RRP Recurrent Respiratory Papillomatosis
  • HPV-11 the viruses can also be detected in the oropharynx of over 50% of normal school-age children and so host factors are clearly important. It is postulated that HPV transmission is mediated during transit through the birth canal in juvenile onset RRP and during oro-genital contact in adult onset RRP. Patients with the disease have no obvious immune deficit.
  • VESPA a new method for virus typing that is referred to hereinbelow as "Viral Evaluation using Self-Probing Amplicons” (VESPA).
  • VESPA is a real-time PCR-based technique that uses self-probing amplicon primers, which are described by Whitcombe et al in Nat Biotechnol 17 804-7 (1999).
  • Such self-probing amplicon primers comprise a specific probe sequence that is held in a hairpin loop configuration by complementary stem sequences on the 5' and 3" sides of the probe.
  • a fluorophore such as 6-carboxyfluorescein
  • a moiety such as methyl red
  • PCR stopper or blocker. After extension of the primer during PCR amplification, the specific probe sequence is able to bind to its complement within the same strand of DNA. This hybridisation event opens the hairpin loop, so that fluorescence is no longer quenched and an increase in signal is observed.
  • the PCR stopper on the primer prevents read-through to the probe, which could lead to opening of the hairpin loop in the absence of the specific target sequence. Such read-through would lead to the detection of non-specific PCR products, eg primer dimers or mis-priming events.
  • the term "self-probing amplicon” refers to a molecule comprising a primer component, a probe component and a signalling system (which may comprise the fluorophore/quencher system as described above or an alternative), as described above and by Whitcombe (1999) (q.v.).
  • a signalling system which may comprise the fluorophore/quencher system as described above or an alternative
  • Whitcombe (1999) (q.v.) q.v.
  • the self-probing amplicon system does not require a separate probe.
  • the self- probing amplicons work in a unimolecular manner, leading to advantages both in terms of simplicity and signal to noise ratios when compared to the bi-molecular probing of 'molecular beacons' and TaqMan.
  • self-probing amplicons can be adapted to mutation or allelic discrimination by monitoring the fluorescence at a temperature where the probe has dissociated from a target with a mismatch but remains bound to a complementary target. This is different from the allelic discrimination by self-probing amplicons described by Whitcombe et al, as they used the ARMS system, whereby the primer rather than the probe is sited over the polymorphic site.
  • VESPA methodology is well suited to virus detection since it is simple to perform, rapid, highly specific, sensitive, reproducible, and has the potential to measure viral load and integration status.
  • the method of the invention has been tested and it has produced typing results on 108 samples, including cell lines, cervical cytobrush samples and tumour biopsies, and preliminary viral load per cell data on 16 clinically-defined samples has been obtained, as described hereinbelow in the Examples.
  • Hybrid Capture IITM and PCR-EIA VESPA is technically less demanding and able to produce results more rapidly.
  • the sensitivity of VESPA in cell lines is at least two orders of magnitude better than that reported by Digene (5000 copies) for Hybrid Capture IITM (from Digene Corporation,
  • VESPA has comparable sensitivity to other previously-published HPV detection techniques, including PCR-EIA (ie in the range of from 1 to 100 viral copies), in which the detection limit is calculated using enriched control targets against a low background of genomic DNA.
  • PCR-EIA ie in the range of from 1 to 100 viral copies
  • the exact viral threshold for immediate risk of carcinogenesis is controversial (and may vary with HPV type and patient), but is likely to be well above VESPA's lower detection limit.
  • VESPA has the potential to estimate viral load (ie per cell rather than gross amount of virus present).
  • viral load per cell is a critical determinant in patient prognosis. Indeed, the so-called "high-risk" types may not be more potent due to the increased oncogenicity of their transforming proteins, for example, but simply because they proliferate more efficiently, overwhelming the immune response.
  • the present invention further provides a degenerate virus self-probing amplicon mix for use in conjunction with a tailed general primer and, optionally, viral specific reverse primers that identify any one or more of the HPV viruses but, most ideally, viral specific reverse primers that are engineered to hybridise to a consensus site within the high-risk HPV types or the low-risk HPV types.
  • a degenerate virus self-probing amplicon can be used with a tailed general primer and a viral specific reverse primer in order to determine, in a sample, the type of HPV infection regardless of the fact that the virus self-probing amplicon is degenerate.
  • tailed primer By using a tailed primer, it is possible to introduce a consensus site that enables a single self-probing amplicon to recognise many different virus amplification products.
  • the tailed primer and degenerate amplicon combination can detect over forty different HPV types.
  • using a viral specific reverse primer enables viral types to be identified.
  • viral DNA is amplified using the prior art consensus primers (GP5+/GP6+), then analysed by ethidium bromide staining of electrophoresed agarose gels. If an amplification product is observed, then it is typed by ELISA using type-specific probes. In practice, however, all samples are typed by
  • VESPA could circumvent this problem by providing improved sensitivity at the pre-typing stage.
  • the concept of a tailed primer is known, the use of the tail as a primer site and the primer as a probe- binding site for a self-probing amplicon is new.
  • the method of the invention for the characterisation of viruses such as human papillomavirus is quicker ( ⁇ 1 hour); more specific (single base discrimination); and less laborious (single step) than currently available techniques and, unlike most techniques, is capable of estimating viral load per cell. It can also be used to determine integration status of the virus. Especially important is the ability of the technique of the invention to determine a plurality of virus types by using tailed primers.
  • Figure 1 relates to HPV-16 detection by the method of the invention, in which: Figure 1a shows the results of HPV typing reactions using self-probing amplicon primers specific for HPV-16 (Sc16), a positive control (HeLa for HPV-18); a negative control (no DNA); and DNA extracted from a HPV 16 specific cell line (Caski); and
  • Figure 1 b shows the results of HPV typing reactions using the self-probing amplicon primers specific for HPV-18 (Sc18); a positive control (Caski for HPV- 16), a negative control (no DNA); and DNA extracted from an HPV-18 containing cell line (HeLa).
  • Figure 2 relates to examples of positive traces produced by VESPA and shows the results of HPV typing experiments using clinical samples previously typed using PCR-EIA. Primers specific for HPV-6 (Sc6) are shown in Figure 2a, HPV-
  • Figure 3 demonstrates the quantitative nature of VESPA for HPV-16. It shows an HPV-16 dilution series using Sc16. A dilution series of SiHa cells was made from 50,000 cells per reaction to 1 cell per reaction.
  • Figure 4 demonstrates the quantitative nature of VESPA for human beta-globin using ScBG and the dilution series as for Figure 3.
  • Figure 5 is a graph showing estimation of viral load using VESPA.
  • Figure 6 is a diagrammatic representation of a strategy for virus, such as degenerate HPV, detection using VESPA.
  • Figure 7 demonstrates the ability of the degenerate self-probing amplicon mix to detect HPV types -6, -16 and -18.
  • Figures 8 and 9 are schematic representations of virus, such as HPV, typing using self-probing amplicons, in which figure 8 is a diagram of a self-probing amplicon and Figure 9 shows the extension and binding stages of the self- probing amplicon with respect to the viral DNA.
  • Figure 10 is a schematic representation of a strategy for determining integration state of ca virus, using the method according to the invention.
  • Figure 11 is a schematic illustration of a VESPA HPV typing kit. On the left- hand side is shown the methodology that is undertaken in respect of a cervical smear sample and, on the right-hand side, is shown the methodology that is undertaken in respect of a control where water is simply added to the relevant tubes. This schematic is discussed in greater detail hereinafter.
  • Figure 12 shows the primer target, probe target and reverse primer target sites for the various viral types to be investigated using the methodology of the invention.
  • the present invention therefore, provides a virus self-probing amplicon comprising (i) a virus primer capable of hybridising to at least one target viral nucleic acid sequence and undergoing amplification thereof under primer amplification conditions to form a virus primer extension product; (ii) a virus probe comprising a nucleic acid sequence complementary to a target sequence of the virus primer extension product and capable of hybridisation thereto, provided that the self-probing amplicon is adapted to ensure that the virus probe is unresponsive to amplification under the primer amplification conditions; and (iii) a member of a virus signalling system, which system is capable of causing a detectable signal to be effected on hybridisation of the virus probe sequence to the virus primer extension product, whereby presence or absence of the target viral nucleic acid sequence in the sample is indicated by the detectable signal.
  • the target virus nucleic acid sequence is preferably one that is capable of indicating the presence of a virus that is associated with a disease or clinical condition in an animal, especially a mammal, more especially man. Particularly preferred is when the virus is human HPV.
  • the virus is selected from one or more of HPV types 6, 11 , 16, 18, 31 , 33, 39, 40, 42, 43, 44, 45, 51 , 52, 56, 58, 59, 66 and 68.
  • the self-probing amplicon is suitably one having a sequence selected from SEQ ID NOs 1 to 6, corresponding to self-probing virus amplicons named Sc16E1, Sc16E2 and Sc16E6, respectively, where Sc16 refers to the amplicon for self-probing HPV type 16 (refer to Table 6 in Example 4 hereinbelow).
  • the probe is the probe component of the above-noted sequences, namely a sequence selected from SEQ ID Nos 2, 4 or 6.
  • the primer is the primer component of the above-noted sequences, namely, a sequence selected from SEQ ID NOs 1 , 3 or 5.
  • the virus self-probing amplicon comprises a virus primer, as afore described, represented by SEQ ID No 1 ; a virus probe, as afore described, represented by SEQ ID No 2 and a virus signalling mechanism which is, preferably, a light emitting mechanism and, ideally, a chemiluminescent molecule and a corresponding quencher molecule.
  • the virus cell probing amplicon comprises a virus primer, as afore described, represented by SEQ ID No 3; a virus probe, as afore described, represented by SEQ ID No 4 and a virus signalling mechanism which is, preferably, a light emitting mechanism and, ideally, a chemiluminescent molecule and a corresponding quencher molecule.
  • the virus cell probing amplicon comprises a virus primer, as afore described, represented by SEQ ID No 5; a virus probe, as afore described, represented by SEQ ID No 6 and a virus signalling mechanism which is, preferably, a light emitting mechanism and, ideally, a chemiluminescent molecule and a corresponding quencher molecule.
  • the 'viral self-probing amplicons' include those that allow detection of changes in the E1 , E2, E6 and/or E7 HPV genes in order to determine integration state of the viral genome in the sample cell genome. Integration status can be measured using two different assessment methods: (i) Ratio of circular viral DNA to linear viral DNA; and (ii) Ratio of cell cycle control viral proteins to cell transforming proteins.
  • the second of these methods relies on the fact that, upon integration of viral DNA into the host genome, the section of viral DNA responsible for the control of viral DNA replication and translation into proteins is excised. This allows the proteins known to be responsible for carcinogenesis to replicate out of control.
  • E1 and E2 the DNA ratio of the control proteins
  • E6 and E7 the transforming proteins
  • the virus primer of the self-probing amplicon is designed to hybridise to at least one high-risk target viral nucleic acid.
  • High-risk viral types have been described hereinbefore (see page 5). High-risk viruses tend to lead to the development of a disease condition and, in the worst instance, cancer.
  • These viruses are characterised by the production of E6 proteins wherein, at the carboxy terminus of the E6 protein, there is a protein motif called the PDZ domain (proteasomal degradation zone). This protein motif is defined as -X-T-X-V (where X is any amino acid, T is threonine and V is valine). This protein motif is therefore a useful tag for discriminating between high and low risk viral types and can therefore be used as a target site for the virus primer of the self-probing amplicon.
  • this motif enables the HPV E6 protein to bind to tumour suppressor proteins. In HPV infected cells this motif therefore no longer functions as a PDZ degradation signal and, moreover, it inactivates the tumour suppressor protein. For these, non-limiting, reasons it is thought that this motif characterises high-risk viral types.
  • Figure 12 shows primer target sites within a range of viruses, including high-risk viruses, and also corresponding probe target sites. Moreover, there is shown reverse primer target sites for each of the relevant viruses.
  • the self-probing amplicon will contain a virus primer that is specific for the region of the nucleic acid molecule encoding the aforementioned motif and, ideally, a degenerate virus probe.
  • the self-probing amplicon may contain a degenerate virus primer and a virus probe that is specific for the nucleic acid encoding the afore motif and so specific for a high- risk virus.
  • the self-probing amplicon primer and probe may both be degenerate and it is the reverse primer that is specific for a high-risk virus.
  • a self-probing amplicon of the invention wherein the target comprises more than one nucleic acid sequence from more than one virus and/or the virus primer component exhibits some degeneracy with respect to the target, whereby the virus primer is not entirely complementary to each one of the nucleic acid sequences of the target.
  • the target comprises more than one nucleic acid sequence from more than one virus and/or the virus primer component exhibits some degeneracy with respect to the target, whereby the virus primer is not entirely complementary to each one of the nucleic acid sequences of the target.
  • Such a preliminary assay may comprise any previously known method for virus detection, including those mentioned hereinbefore.
  • the preliminary assay comprises the method of the invention wherein a tailed primer is first incorporated into the viral primer extension product.
  • the tail comprises a nucleic acid sequence capable of amplifying, eg under PCR conditions, the viral nucleic acid sequence of a plurality of viruses or virus, such as HPV, types. It is believed that this is the first time the concept of tailed primers has been applied to self-probing amplicons.
  • a unique self-probing amplicon target site is introduced into the amplification (primer extension) product.
  • a unique 'designer tail' is attached to the prior art consensus primers (such as one capable of amplifying all 20 of the common HPV types)
  • this aim can be achieved.
  • a preferred tailed primer for use in the present invention is shown in Table 7 of
  • Example 5 [SEQ ID NO: 7]. Two suitable degenerate self-probing amplicons for use in the present invention are shown in Table 7 of Example 5 [SEQ ID NOs: 8 & 9]; these sequences also comprise preferred primer and probe components. This methodology is illustrated in Figure 6 where it can be seen that a tailed primer, wherein the tail contains the designer sequence ATG TGG AAA CAT GCA TGG, is integrated into viral DNA using two rounds of PCR amplification. Moreover, it follows that if the reverse primer is specific for a particular HPV or group of HPV's then the tailed primer will be integrated into a specific type of HPV.
  • the present invention further provides the novel tail part [SEQ ID NO: 10] of these sequences; the probe part of these sequences being designed to bind to the known GP6+ sequence.
  • the present invention also provides a diagnostic kit for use in a method of the invention, which kit comprises one or more of the virus self-probing amplicons, housekeeping or control self-probing amplicons or tailed primers of the invention.
  • kit suitable for use in determining viral load per cell, and accordingly comprises at least two, and suitably four or more, self- probing amplicons, including at least one housekeeping self-probing amplicon and, optionally, a tailed primer and/or a reverse primer that is specific for a particular type of HPV or group of HPV's such as high-risk HPV's or low risk HPV's.
  • kits comprising at least one self-probing amplicon for targeting at least one of the E1 , E2, E6 and E7 HPV genes.
  • the reverse primer is specific for a high-risk HPV, it is designed to, ideally, bind to the part of the nucleic acid that encodes the PDZ domain of the E6 protein and so is designed to bind to the target sites, for the high-risk viral types, shown in Figure 12.
  • the invention provides the use of such self-probing amplicons, including the probe and primer components thereof, in a method as per below or in the preparation of a kit as hereinbefore described.
  • the invention further provides a method for one or more of:
  • a self-probing amplicon irus self-probing amplicon'
  • a virus primer capable of hybridising to at least one target viral nucleic acid sequence and undergoing amplification thereof under primer amplification conditions to form a virus primer extension product
  • a virus probe comprising a nucleic acid sequence complementary to a target sequence of the virus primer extension product and capable of hybridisation thereto, provided that the self-probing amplicon is adapted to ensure that the virus probe is unresponsive to amplification under the primer amplification conditions
  • a member of a virus signalling system which system is capable of causing a detectable signal to be effected on hybridisation of the virus probe sequence to the virus primer extension product, whereby presence or absence of the target viral nucleic acid sequence in the sample is indicated by the detectable signal; and, optionally,
  • step (IB) amplifying the product of step (IA) under the primer amplification conditions to an extent enabling the detectable signal to be effected after step (II); then
  • (II) separating the virus primer extension product from the target viral nucleic acid sequence; allowing the virus probe to hybridise to the target sequence of the virus primer extension product; and monitoring the signalling system, wherein the 'viral self-probing amplicon(s)' is/are adapted to allow detection, quantification or assessment of the E1 , E2, E6 and/or E7 HPV genes.
  • the invention further provides a method comprising the following steps:
  • (0)(A) contacting a target viral nucleic acid sequence from the sample with 'tailed primer' which comprises: (i) a primer region comprising a nucleic acid sequence ('consensus primer sequence') complementary to a consensus sequence of the viral nucleic acid sequence and capable of hybridisation thereto and undergoing amplification thereof under primer amplification conditions to form a tailed primer extension product; and (ii) a tail region comprising a unique sequence not present in or prepared by any component of this method ('designer' sequence); and
  • the primer component of the 'virus self-probing amplicon' is capable of binding to the 'designer' sequence and the probe component of the virus self- probing amplicon is complementary to the consensus primer sequence.
  • said virus primer of the self-probing amplicon hybridises to at least one target high-risk viral nucleic acid and so, ideally, includes a nucleic acid sequence that is capable of hybridising to nucleic acid encoding a motif, as herein described, that characterises a high-risk virus or to a primer target site shown in Figure 12.
  • the virus probe of the self-probing amplicon hybridises to at least one target high-risk viral nucleic acid as afore described.
  • the reverse primer is designed to hybridise to nucleic acid encoding a motif, as herein described, that characterises a high-risk virus or to the reverse primer target 3' - 5' antisense sequence shown in Table 12.
  • DNA was purified from cell lines by re-suspension of cells in 640 I of Nuclear Lysis Buffer (10mM Tris HCI, 0.4M NaCl, 2mM ethylenediamine tetra-acetate pH 8.0, 10% sodium dodecyl sulphate), 100 I of 6M NaCl and 740 I of chloroform. The solution was thoroughly mixed, centrifuged and the top phase extracted. DNA was precipitated by the addition of 1ml 95% ethanol and pelleted by centrifugation. The pellet was washed twice with 70% ethanol, dried in a rotary evaporator and re-suspended in 500 I of de-ionised water.
  • Nuclear Lysis Buffer 10mM Tris HCI, 0.4M NaCl, 2mM ethylenediamine tetra-acetate pH 8.0, 10% sodium dodecyl sulphate
  • DNA was purified from cervical brush samples by a simple modification (squeezing the cytobrushes on the side of the tube, and freezing times were increased to 24 hours from 2 hours) of the freeze-thaw method as described by Jacobs et al in Jacobs et al J Clin Microbiol 35 791-5 (1997).
  • Epithelial cells obtained from the cytobrush samples were pelleted by centrifugation and re-suspended in 1ml 10mM Tris pH 7.4, and frozen at -70°C for 24 hours. A 100 I aliquot was thawed, boiled for 10 minutes; chilled on ice; spun at 13,000 rpm in a microfuge for 3 minutes; and supernatant decanted and stored.
  • DNA was extracted from biopsy material by incubation in 1 ml 10mM Tris HCI pH7.4 containing 10mg/ml proteinase K (from Sigma, UK) for 1 hour at 56°C before boiling to activate the enzyme.
  • PCR-EIA was performed as described by Jacobs et al (q.v.).
  • Table 6 shows the sequences of the six self-probing amplicon primers used in this study. All primers were synthesised by Oswel Research Products, Southampton S016 7PX, UK.
  • All self-probing amplicon primers were purified by double HPLC. Standard primers were purified by gel filtration. Primers were designed to detect the HPV L1 gene. Primer locations varied between types but were positioned approximately between 6600 and 6750 bp. The forward primer sequence of each self-probing amplicon is type-specific, and is located at the same sequence position as that of the GP6 + primer described by Jacobs et al (q.v.). Self-probing amplicon probe sequences were designed by aligning the L1 open reading frames (ORF) of twenty common HPV types (HPV-6, 11 , 16, 18, 31 , 33, 39, 40, 42, 43, 44, 45, 51 , 52, 56, 58, 59, 66 and 68)
  • the SC16 primer component comprises the known GP6+ sequence.
  • the reverse primer target sequence is the GP5 + sequence of Jacobs et al (q.v.).
  • the HPV 16 and 18 primers were tested for specificity using reference cell lines with integrated HPV DNA.
  • the Caski cell line contains 60-600 copies of the
  • HPV-16 ORF per cell and the HeLa cell line contains 10-50 copies of the HPV- 18 L1 ORF.
  • Figure 1a shows the results of PCR reactions using the Sc16 self-probing amplicon primer (designed for detection of HPV-16 DNA), and DNA extracted from the HPV-16 positive Caski cell line, DNA from the HPV-18 positive HeLa cell line and a negative control (no DNA). A significant increase in fluorescence was only detected with the HPV-16 containing Caski DNA.
  • Figure 1 b shows a similar experiment using the Sc18 self-probing amplicon primer in place of Sc16. Here, significant fluorescence was only detected with the HPV-18 positive HeLa cell line. The primers were then used to detect HPV- 16 and -18 in clinical samples previously typed using PCR-EIA ( Figure 2c and Figure 2d - see below).
  • a theoretical advantage of self-probing amplicon PCR is its ability to determine viral load. Shown, in Figure 3, are the results of Sc16 typing reactions performed using a dilution series of the SiHa cell line (one to two copies of HPV- 16 per cell). The dilution series from 50,000 to 500 HPV copies per cell were clearly distinguishable, and the signal for HPV-16 remained positive in the sample containing a single copy of HPV-16 DNA.
  • a primer was designed to detect (human) beta- globin DNA (ScBG, SEQ ID NO: 11 in Table 3).
  • ScBG beta- globin DNA
  • Table 3 - HPV-16 Viral Load - Sequences of VESPA Primers.
  • Figure 4 shows the results of an experiment conducted using a self-probing amplicon designed to detect the human beta-globin gene over the same dilution series of SiHa cells used above for HPV-16. Once again, the signal remained positive down to a single cell and is quantitative at and above 50 copies per cell.
  • Figure 5 shows a plot of the ratio of the fluorescence produced by Sc16 and ScBG against the logarithm of the viral copy number per cell of target DNA. A range of copy numbers per cell may be calculated from a cell line containing a fixed copy number, by calculating the ratio of two different dilutions.
  • the Sc16 Fm a x value for 5000 cells can be divided by that for the ScBG Fm a x for 100 cells to obtain a value for a notional cell line containing 50 copies per cell (5000/100).
  • Disease grade was determined by cytology.
  • VL ratio (Viral Load) was measured in RSU (Relative Self-probing amplicon Units) by dividing Sc16 Fm a x by ScBG Fmax- Viral copies per cell were estimated using the standard curve shown in Figure 5.
  • PCR- EIA results produced by PCR- EIA might be false positives. There is evidence from the study comparing PCR- EIA in several different laboratories that it is prone to the occasional false positive. The fact that four of the six discrepant samples have normal cytology supports this supposition. Of the remaining two, one has low-grade lesions and for the other we have no clinical data. Secondly, the samples may be positive for HPV but contain an intra-typic HPV variant containing polymorphism within the probe-binding site.
  • PCR-EIA probes discriminate between sequences on the basis of a single base change, whereas PCR-EIA probes are more tolerant of sequence variation. In this regard, it may be relevant that 5/6 discordant samples were obtained from West Africa, where there is likely to be more variation within the probe binding site. Table 5 - Comparison of PCR-EIA and VESPA
  • Self-probing amplicon sequences designed for the assessment of HPV integration state are shown in Table 6. They are used in accordance with the method described in the Examples above. Integration status is measured as described hereinabove.
  • Sc self-probing amplicon
  • RP reverse primer
  • Bold typeface indicates probe and primer sequences, respectively.
  • a suitable strategy comprises the following steps, illustrated by Figure 6: 1.
  • a primer containing a 'designer tail' is used to amplify viral DNA (if present).
  • a standard reverse primer is also included.
  • a HPV specific reverse primer may be included so ensuring that the "designer tail" is integrated into a specific type of HPV and so making the following methodology specific for the detection of a specific type of HPV.
  • a HPV specific reverse primer may be engineered so that it identifies one or more HPV types and so a target group of HPV viruses may be detected.
  • any one or more HPV specific reverse primers may be used so as to target the nature of the HPV nucleic acid that is to be detected.
  • Amplification proceeds as in a standard reaction.
  • the 'designer' sequence has no complementary sequence.
  • this amplification can then be detected using a self-probing amplicon comprising a primer targeting the 'designer' sequence and a probe for detecting the consensus primer. It may be necessary to use two self-probing amplicons with a slight overhang into the viral DNA to avoid unacceptable background fluorescence caused by primer dimer.
  • Figure 11 is a schematic illustration of the methodology that is described above.
  • Tube 1 contains at least one degenerate Scorpion or virus self-probing amplicon with a virus primer capable of hybridising to "designer sequence”; a tailed primer wherein the tail comprises a "designer sequence” and one or more reverse primers that are designed to hybridise to a region of a high-risk HPV, plus appropriate reagents.
  • Tube 2 contains a degenerate Scorpion or virus self-probing amplicon with a virus primer capable of hybridising to a "designer sequence”; a tailed primer wherein the tail comprises a "designer sequence” and at least one reverse primer that is specific for at least one low-risk HPV, plus appropriate reagents. If HPV is present in the cervical smear sample then, if the HPV is a high-risk HPV, the tailed primer and the reverse primer with the high-risk specific sequence will bind thereto and amplification will take place resulting in the "designer sequence" being incorporated into the viral DNA.
  • the virus self- probing amplicon will bind, via its virus primer, to the "designer sequence" that has been incorporated into the viral DNA and the virus self-probing amplicon will therefore operate in such a fashion that a primer extension is produced and the virus signalling system will be activated resulting in a signal, typically a fluorescent signal, being emitted.
  • a fluorescent signal will signify the presence of high-risk HPV in the original sample.
  • Tube 3 has the same contents as Tube 1 and
  • Tube 4 has the same contents as Tube 2. Given that water is added to Tubes 3 and 4, there should be no HPV in the water sample and therefore no fluorescent signal should be produced.
  • Sc self-probing amplicon
  • Figure 7 demonstrates the ability of the degenerate self-probing amplicon mix to detect HPV types -6, -16 and -18. Comparisons may be performed using samples pre-amplified under conditions described by Jacobs et al (qv). Interestingly, two of these samples were barely visible after agarose gel electrophoresis with ethidium bromide staining, suggesting that the VESPA approach produces the expected improvement in sensitivity.
  • Pre-amplification First Round Amplification
  • Each reaction was set up three times with appropriate primer pairs in order to test for SV40, JC and BK.
  • each sample per reaction was pre-amplified under the following conditions; 0.5 M P1 and P3 (see Table 8), 2.5mM MgCI 2 , 200 M dNTPs, 1.25U of Taq polymerase (Advanced Biotechnologies, Epsom, Surrey, UK) and 5 I of 10X reaction buffer (Perkin-Elmer Gold) in a total reaction volume of 50 I.
  • Each sample was also SV40-typed under the conditions described above and using the P1-P2 primer pair (Table 8).
  • Assay set-up was performed in a laboratory physically separated (100m) from that used for the first round amplification.
  • PCR amplification of 1 I aliquots of DNA (2 I iCycler) solution were performed using 0.5 M Scorpion primer (see Table 1) and 0.5 M reverse primer in a total reaction volume of 10 I (20 I iCycler). Reaction conditions were as follows: 200 M dNTPs, 4mM MgCI 2 , 50mM Tris HCI pH 8.9, 10mM ammonium sulphate, 0.1 % Tween 20, bovine serum albumin 250ng/ I, and 0.5 U/ I Taq polymerase (Advanced Biotechnologies, Epsom, Surrey, UK). Fluorescence was detected in channel one/530nm (FilterSet4 iCycler) and at 40°C (45°C iCycler).
  • Self-probing amplicon control reaction contained H 2 0 in place of DNA. All primers were synthesised by Oswel Research Products, Southampton S016 7PX, UK. Cycling Parameters Pre-amplification 94°C for 180s followed by 30 cycles of 94°C-60s, 52°C-60s and 72°C-60s and finally 72°C-300s.
  • Amplicon Detection i) LightCycler. 100 cycles of 96°C-1 s, 40° C-5s, 72° C-1 s. (ii) iCycler. 95°C for 180s followed by 40 cycles of 95°C-30s, 45°C-30s,
  • RRP respiratory papillomatosis
  • the viral loads presented in Table 10 were calculated as described in Example 2. Briefly, the ratio of the fluorescence signals produced using a cognate HPV- self-probing amplicon and that of a self-probing amplicon designed to detect a human genomic beta-globin housekeeping gene was calculated.
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Abstract

This invention relates to a method and means for detecting, typing and characterising the presence of viruses in a tissue sample and in particular the invention relates to a novel light-emitting self-probing amplicon that comprises a primer, probe and signalling system.

Description

VIRUS DETECTION METHOD, PRIMERS THEREFOR AND SCREENING KIT
The present invention relates to an improved method for detecting, typing and characterising the presence of viruses, particularly those that have been associated with carcinogenic activity in mammals, such as human papillomavirus, particularly, but not exclusively, in cervical samples and to primers and probes for use in the method. The invention further relates to a diagnostic kit and screening method, which uses the kit.
As well as there being various viruses that have been associated with carcinogenic effects and therefore there being a need to be able to identify the presence of such viruses in clinical samples, it is also important to be able to accurately determine the viral load per cell and integration status of the virus in the affected organism (most importantly, in humans). Accordingly, it is desired to provide means for establishing the infecting virus type, the viral load (per cell ie the number of viral copies per cell or ratio of viral genome : human genome) and the integration state (ie whether the viral DNA is 'free' in the cytosol of the cell or integrated into the host genome) of the virus, since these factors are known to have profound implications for patient prognosis.
Cervical cancer is the second most frequent cause of death from cancer in women, worldwide. Cervical screening programmes reduce the incidence of cervical cancer; however, 50% of invasive cervical cancers arise in women screened using existing cytological methodologies. In recent years, it has been established that a subset of human papillomaviruses (HPV) are associated with cervical cancer, and it is estimated that HPV DNA is present in over 99% of these cancers. There are currently 84 types of HPV, around 30 of which infect the genital tract. Therefore, HPV detection and typing techniques have been proposed as an adjunct to, or replacement for, the current cytological screening regime. Clearly, the success of such strategies will depend on the development of rapid, reliable, sensitive and specific HPV-detection methods applicable in the clinical setting.
Currently, there are eight main approaches to the detection and typing of HPV, all of which display advantages and disadvantages, depending on application. These are summarised in Table 1 , from which it can be seen that no single technique performs optimally in both clinical and research settings. By way of background, the PCR method has been introduced as the most sensitive method for the detection of HPV DNA in clinical specimens. However, a significant heterogeneity at the nucleotide level is found between the different HPV genotypes. This has hampered the development of a simple, universal PCR test for the detection of all HPV genotypes. Despite this, HPV PCR methods have been developed, allowing the detection of a broad spectrum of mainly mucosotropic HPV genotypes. Table 1 : Current HPV Detection and Typing Techniques.
Figure imgf000004_0001
HPV is genetically composed of early (E) and late (L) genes, which are functionally divided into several open reading frames (ORFs): viral replication (E1), regulation of transcription (E2), coding for cytoplasmic proteins (E4) and malignant transformation (E5, E6, E7) as early genes, and coding for capsid proteins (L1 , L2) as late genes.
A combination of the general primers GP5 and GP6, originally selected from the HPV L1 region on the basis of sequence information from HPV-6, -11 , -16, -18, - 31 and -33, was found to amplify target DNA of at least 27 mucosotropic HPV genotypes under conditions that allow mismatch acceptance. The general primers GP5 (δ'-TTT GTT ACT GTG GTA GAT AC-3') and GP6 (3'-ACT AAA TGT CAA ATA AAA AG-5') (Snijders et al, J.Gen. Virol. 71 173-181 (1990)), which span a region of 140-150 bp from the L1 open reading frame of a broad spectrum of HPV genotypes, were used in general primer-mediated PCR. The strength of this (GP-PCR) method has been further substantiated by the detection of HPV DNA in 100% of cervical scrapes cytomorphologically classified as Pap IV (carcinoma in situ) and Pap V (carcinoma) in the Netherlands. This suggests that, in the Dutch population, all genital high-risk HPV's can be detected by this assay.
But, using GP-PCR in routine diagnostic practice, it has been found that a small number of clinical samples give rise to ambiguous results, reflected by weak GP- PCR signals accompanied by a relatively high background of co-amplified cellular DNA. It was then found that increased primer length contributes to more efficient amplification, and so the GP5 and GP6 primers were elongated with highly conserved sequences at the 3' ends. When the use of these GP5+ and GP6+ primers in the PCR was compared with the original GP5 and GP6 PCR on Ing cloned DNA of various HPV types, it was found that the elongated general primers were significantly more sensitive.
In order to facilitate PCR-based HPV detection and typing, a colorimetric microtitre plate based hybridisation assay was developed. The method used one biotinylated primer (bio-GP6+) in the GP-PCR. Biotinylated PCR products were captured on streptavidin-coated microtitre plates, denaturated and hybridised to digoxigenin-(DIG-) labelled HPV-specific internal oligonucleotide probes. The DIG-labelled hybrids were detected using an enzyme immunoassay (EIA). In a development of this technique, two cocktails of DIG- labelled HPV type-specific oligonucleotide probes and an EIA have been used as a basis to develop a group-specific detection method for 14 high-risk (types
16, 18, 31 , 35, 39, 45, 51 , 52, 56, 58, 59, 66 and 68) and 6 low-risk (types 6, 11 , 40, 42, 43 and 44) HPVs following a general primer GP5+/bioGP6+-mediated PCR. This technique is hereinafter referred to as PCR-EIA. The sensitivity of this high-risk/low risk (HR/LR) HPV PCR-EIA ranged from 10 to 200 HPV copies, depending on the HPV type.
Some fluorogenic probe assays for HPV DNAs are based on the PCR amplification of a portion of the L1 open reading frames of HPV-16, -18, -31 , -33, and -35 DNAs by using genotype-specific probes that bind to the amplified DNA. In the TaqMan assay, the probes are blocked at their 3' termini and hence cannot be extended by the polymerase. If, during the course of primer extension, Taq polymerase encounters a bound probe, its 5'- 3' exonuclease activity degrades the probe, releasing the 5' fluor from the 3' quencher. This causes an increase in the fluorescence emitted by the reporter which, in the presence of an excess amount of the probe, is directly related to the amount of
HPV DNA present in the sample before amplification. 'Molecular beacons' comprise a probe flanked by a hairpin loop that holds a fluorophore and quencher in close proximity until specific binding of the probe to its target opens out the structure, producing a fluorescent signal. In general, HPV DNA is present as an episomal form in low grade cervical intra- epithelial neoplasia (CIN) lesions, and the principal form of the viral DNA in high grade CIN and invasive cancers is integrated into the host genome. In most cervical carcinomas, HPV genomes are integrated into host cell chromosomes and transcribed into mRNAs encompassing viral and cellular sequences. In contrast, in early pre-neoplastic lesions, HPV genomes persist as episomes, and derived transcripts contain exclusively viral sequences. Thus, detection of HPV transcripts derived from integrated HPV genomes may specifically indicate both CIN lesions at high-risk for progression as well as invasive cervical cancers.
When the HPV DNA becomes integrated into the cellular chromosome, generally some part of the E2 ORF is lost, releasing suppression of E6 and E7 mRNA expression. The E6 and E7 proteins can activate some oncogenes and/or inactivate host tumour suppressor gene products (p53, pRB), resulting in uncontrolled cellular growth and malignant transformation.
Integration of HPV DNA is therefore considered an important genetic change in the process of cervical carcinogenesis, and being able to determine the level of integration is believed to be important, diagnostically. Some detection techniques therefore focus on identifying and quantifying disruptions in the early genes, particularly E1/E2 and E6/E7. These have been carried out using techniques such as multiplex PCR (where primers for each sequence are included in the same reaction) and RT-PCR that allows discrimination of HPV mRNAs derived from integrated and episomal viral genomes.
Hitherto-known techniques therefore require a plurality of techniques and/or probes to detect HPV types and integration status, which is time-consuming, slow and generally unsatisfactory for clinical use. Furthermore, none of these techniques satisfactorily deal with the determination of viral load, since none result in a measure of the number of viral copies per cell. These systems have used different methods for the quantitation of virus copy number and cell copy number and thus the two are not strictly comparable. Previous methods incorporating assays for the β-globin or other housekeeping genes of the genomic DNA have, at most, given rise to a range of viral DNA copies per microgram of cellular DNA, rather than per cell. Other uses of housekeeping genes have been as PCR controls or to determine PCR limits and not to provide information about viral load.
Another disease caused by HPV is Recurrent Respiratory Papillomatosis (RRP), which is the most common benign tumour of the larynx, and is a serious condition with no satisfactory treatment and around 2-7% malignant transformation. RRP is caused by one of two HP viruses, namely, HPV-6 and
HPV-11. However, these viruses can also be detected in the oropharynx of over 50% of normal school-age children and so host factors are clearly important. It is postulated that HPV transmission is mediated during transit through the birth canal in juvenile onset RRP and during oro-genital contact in adult onset RRP. Patients with the disease have no obvious immune deficit.
Accordingly, the present invention provides a new method for virus typing that is referred to hereinbelow as "Viral Evaluation using Self-Probing Amplicons" (VESPA). VESPA is a real-time PCR-based technique that uses self-probing amplicon primers, which are described by Whitcombe et al in Nat Biotechnol 17 804-7 (1999).
Such self-probing amplicon primers comprise a specific probe sequence that is held in a hairpin loop configuration by complementary stem sequences on the 5' and 3" sides of the probe. A fluorophore (such as 6-carboxyfluorescein) attached to the 5'-end is quenched by a moiety (such as methyl red) joined to the 3'-end of the loop. The hairpin loop is linked to the 5'-end of a primer via a
PCR stopper or blocker. After extension of the primer during PCR amplification, the specific probe sequence is able to bind to its complement within the same strand of DNA. This hybridisation event opens the hairpin loop, so that fluorescence is no longer quenched and an increase in signal is observed. The PCR stopper on the primer prevents read-through to the probe, which could lead to opening of the hairpin loop in the absence of the specific target sequence. Such read-through would lead to the detection of non-specific PCR products, eg primer dimers or mis-priming events. Accordingly, as used herein, the term "self-probing amplicon" refers to a molecule comprising a primer component, a probe component and a signalling system (which may comprise the fluorophore/quencher system as described above or an alternative), as described above and by Whitcombe (1999) (q.v.). Thus, unlike 'molecular beacons' and TaqMan probes that have been used, the self-probing amplicon system does not require a separate probe. The self- probing amplicons work in a unimolecular manner, leading to advantages both in terms of simplicity and signal to noise ratios when compared to the bi-molecular probing of 'molecular beacons' and TaqMan. Furthermore, such self-probing amplicons can be adapted to mutation or allelic discrimination by monitoring the fluorescence at a temperature where the probe has dissociated from a target with a mismatch but remains bound to a complementary target. This is different from the allelic discrimination by self-probing amplicons described by Whitcombe et al, as they used the ARMS system, whereby the primer rather than the probe is sited over the polymorphic site.
VESPA methodology is well suited to virus detection since it is simple to perform, rapid, highly specific, sensitive, reproducible, and has the potential to measure viral load and integration status. The method of the invention has been tested and it has produced typing results on 108 samples, including cell lines, cervical cytobrush samples and tumour biopsies, and preliminary viral load per cell data on 16 clinically-defined samples has been obtained, as described hereinbelow in the Examples. Furthermore, when compared to both prior art front-line HPV-detection and typing methodologies, Hybrid Capture II™ and PCR-EIA, VESPA is technically less demanding and able to produce results more rapidly. The sensitivity of VESPA in cell lines is at least two orders of magnitude better than that reported by Digene (5000 copies) for Hybrid Capture II™ (from Digene Corporation,
Silver Spring, Md.), which has been approved by The Federal Drugs Administration (FDA in the USA) for HPV screening. VESPA has comparable sensitivity to other previously-published HPV detection techniques, including PCR-EIA (ie in the range of from 1 to 100 viral copies), in which the detection limit is calculated using enriched control targets against a low background of genomic DNA. The exact viral threshold for immediate risk of carcinogenesis is controversial (and may vary with HPV type and patient), but is likely to be well above VESPA's lower detection limit.
The presence of inhibitors (eg haemoglobin) that may prevent efficient DNA amplification in cervical samples has been previously reported. Using a standard chloroform extraction based method of DNA purification, we have demonstrated that it is possible to improve the signal produced by VESPA to detect HPV in cervical smear samples only weakly positive when purified by the freeze-thaw technique usually used with PCR-EIA. VESPA is type-specific and compares favourably with Hybrid Capture™, in which samples are only categorised into "Low-risk" and "High-risk" HPV types. The HPV typing achieved using VESPA
correlates well in our hands with data obtained using PCR-EIA (κ=0.89), the
most established HPV typing method. As well as detection and typing, VESPA has the potential to estimate viral load (ie per cell rather than gross amount of virus present). There is increasing evidence that viral load per cell is a critical determinant in patient prognosis. Indeed, the so-called "high-risk" types may not be more potent due to the increased oncogenicity of their transforming proteins, for example, but simply because they proliferate more efficiently, overwhelming the immune response.
In order to expand the capability of VESPA for use in virus screening, the present invention further provides a degenerate virus self-probing amplicon mix for use in conjunction with a tailed general primer and, optionally, viral specific reverse primers that identify any one or more of the HPV viruses but, most ideally, viral specific reverse primers that are engineered to hybridise to a consensus site within the high-risk HPV types or the low-risk HPV types. In this way, a degenerate virus self-probing amplicon can be used with a tailed general primer and a viral specific reverse primer in order to determine, in a sample, the type of HPV infection regardless of the fact that the virus self-probing amplicon is degenerate. By using a tailed primer, it is possible to introduce a consensus site that enables a single self-probing amplicon to recognise many different virus amplification products. The tailed primer and degenerate amplicon combination can detect over forty different HPV types. Further, using a viral specific reverse primer enables viral types to be identified. In the PCR-EIA technique described by Jacobs et al in J Clin Microbiol 35, 791-5 (1997), viral DNA is amplified using the prior art consensus primers (GP5+/GP6+), then analysed by ethidium bromide staining of electrophoresed agarose gels. If an amplification product is observed, then it is typed by ELISA using type-specific probes. In practice, however, all samples are typed by
ELISA, since agarose gel electrophoresis is not sensitive enough to detect poorly amplified DNA. VESPA could circumvent this problem by providing improved sensitivity at the pre-typing stage. Although the concept of a tailed primer is known, the use of the tail as a primer site and the primer as a probe- binding site for a self-probing amplicon is new.
Therefore, the method of the invention for the characterisation of viruses such as human papillomavirus is quicker (<1 hour); more specific (single base discrimination); and less laborious (single step) than currently available techniques and, unlike most techniques, is capable of estimating viral load per cell. It can also be used to determine integration status of the virus. Especially important is the ability of the technique of the invention to determine a plurality of virus types by using tailed primers.
The invention is further described below, with reference to the following Figures:
Figure 1 : relates to HPV-16 detection by the method of the invention, in which: Figure 1a shows the results of HPV typing reactions using self-probing amplicon primers specific for HPV-16 (Sc16), a positive control (HeLa for HPV-18); a negative control (no DNA); and DNA extracted from a HPV 16 specific cell line (Caski); and
Figure 1 b shows the results of HPV typing reactions using the self-probing amplicon primers specific for HPV-18 (Sc18); a positive control (Caski for HPV- 16), a negative control (no DNA); and DNA extracted from an HPV-18 containing cell line (HeLa).
Figure 2 relates to examples of positive traces produced by VESPA and shows the results of HPV typing experiments using clinical samples previously typed using PCR-EIA. Primers specific for HPV-6 (Sc6) are shown in Figure 2a, HPV-
11 (Sc11) in Figure 2b, HPV-16 (Sc16) in 2c, HPV-18 (Sc18) in 2d, HPV-31 (Sc31) in 2e, HPV-33 (Sc33) in 2f, HPV-39 (Sc39) in 2g, and HPV-51 (Sc51) in 2h.
Figure 3 demonstrates the quantitative nature of VESPA for HPV-16. It shows an HPV-16 dilution series using Sc16. A dilution series of SiHa cells was made from 50,000 cells per reaction to 1 cell per reaction.
Figure 4 demonstrates the quantitative nature of VESPA for human beta-globin using ScBG and the dilution series as for Figure 3.
Figure 5 is a graph showing estimation of viral load using VESPA. Figure 6 is a diagrammatic representation of a strategy for virus, such as degenerate HPV, detection using VESPA.
Figure 7 demonstrates the ability of the degenerate self-probing amplicon mix to detect HPV types -6, -16 and -18.
Figures 8 and 9 are schematic representations of virus, such as HPV, typing using self-probing amplicons, in which figure 8 is a diagram of a self-probing amplicon and Figure 9 shows the extension and binding stages of the self- probing amplicon with respect to the viral DNA.
Figure 10 is a schematic representation of a strategy for determining integration state of ca virus, using the method according to the invention.
Figure 11 is a schematic illustration of a VESPA HPV typing kit. On the left- hand side is shown the methodology that is undertaken in respect of a cervical smear sample and, on the right-hand side, is shown the methodology that is undertaken in respect of a control where water is simply added to the relevant tubes. This schematic is discussed in greater detail hereinafter.
Figure 12 shows the primer target, probe target and reverse primer target sites for the various viral types to be investigated using the methodology of the invention. The present invention, therefore, provides a virus self-probing amplicon comprising (i) a virus primer capable of hybridising to at least one target viral nucleic acid sequence and undergoing amplification thereof under primer amplification conditions to form a virus primer extension product; (ii) a virus probe comprising a nucleic acid sequence complementary to a target sequence of the virus primer extension product and capable of hybridisation thereto, provided that the self-probing amplicon is adapted to ensure that the virus probe is unresponsive to amplification under the primer amplification conditions; and (iii) a member of a virus signalling system, which system is capable of causing a detectable signal to be effected on hybridisation of the virus probe sequence to the virus primer extension product, whereby presence or absence of the target viral nucleic acid sequence in the sample is indicated by the detectable signal.
The target virus nucleic acid sequence is preferably one that is capable of indicating the presence of a virus that is associated with a disease or clinical condition in an animal, especially a mammal, more especially man. Particularly preferred is when the virus is human HPV. Preferably, the virus is selected from one or more of HPV types 6, 11 , 16, 18, 31 , 33, 39, 40, 42, 43, 44, 45, 51 , 52, 56, 58, 59, 66 and 68. In such cases, the self-probing amplicon is suitably one having a sequence selected from SEQ ID NOs 1 to 6, corresponding to self-probing virus amplicons named Sc16E1, Sc16E2 and Sc16E6, respectively, where Sc16 refers to the amplicon for self-probing HPV type 16 (refer to Table 6 in Example 4 hereinbelow). Especially preferred is when the probe is the probe component of the above-noted sequences, namely a sequence selected from SEQ ID Nos 2, 4 or 6.
Preferably, the primer is the primer component of the above-noted sequences, namely, a sequence selected from SEQ ID NOs 1 , 3 or 5.
In a preferred embodiment of the invention the virus self-probing amplicon comprises a virus primer, as afore described, represented by SEQ ID No 1 ; a virus probe, as afore described, represented by SEQ ID No 2 and a virus signalling mechanism which is, preferably, a light emitting mechanism and, ideally, a chemiluminescent molecule and a corresponding quencher molecule.
In another preferred embodiment of the invention the virus cell probing amplicon comprises a virus primer, as afore described, represented by SEQ ID No 3; a virus probe, as afore described, represented by SEQ ID No 4 and a virus signalling mechanism which is, preferably, a light emitting mechanism and, ideally, a chemiluminescent molecule and a corresponding quencher molecule. In yet another preferred embodiment of the invention the virus cell probing amplicon comprises a virus primer, as afore described, represented by SEQ ID No 5; a virus probe, as afore described, represented by SEQ ID No 6 and a virus signalling mechanism which is, preferably, a light emitting mechanism and, ideally, a chemiluminescent molecule and a corresponding quencher molecule.
The 'viral self-probing amplicons' include those that allow detection of changes in the E1 , E2, E6 and/or E7 HPV genes in order to determine integration state of the viral genome in the sample cell genome. Integration status can be measured using two different assessment methods: (i) Ratio of circular viral DNA to linear viral DNA; and (ii) Ratio of cell cycle control viral proteins to cell transforming proteins.
The first of these methods (i) is illustrated in Figure 10 (as described by Park et al in Gynecol Oncol 65(1) 121-9 (1997)). Each sample is assessed, using the method of the invention, for the quantitative amount of viral DNA in circular form. This assessment may be achieved by using self-probing amplicons designed to contact the viral DNA before the putative viral DNA break point (in the middle of the E1 protein) and a reverse primer after the putative viral break point (in the E6 or, preferably, E7 protein). This assessment reflects the quantitative amount of viral DNA in circular form. This result may then be compared with total viral DNA, as determined by the method of the invention described above, to give a percentage of viral DNA in circular form. The second of these methods (ii) relies on the fact that, upon integration of viral DNA into the host genome, the section of viral DNA responsible for the control of viral DNA replication and translation into proteins is excised. This allows the proteins known to be responsible for carcinogenesis to replicate out of control. Thus, by quantitatively measuring the DNA ratio of the control proteins (E1 and E2) to the transforming proteins (E6 and E7), it is possible to assess the likelihood that the sample is derived from a patient that is about to undergo malignant transformation.
In yet a further preferred embodiment of the invention the virus primer of the self-probing amplicon is designed to hybridise to at least one high-risk target viral nucleic acid. High-risk viral types have been described hereinbefore (see page 5). High-risk viruses tend to lead to the development of a disease condition and, in the worst instance, cancer. These viruses are characterised by the production of E6 proteins wherein, at the carboxy terminus of the E6 protein, there is a protein motif called the PDZ domain (proteasomal degradation zone). This protein motif is defined as -X-T-X-V (where X is any amino acid, T is threonine and V is valine). This protein motif is therefore a useful tag for discriminating between high and low risk viral types and can therefore be used as a target site for the virus primer of the self-probing amplicon.
It is thought that the functionality of this motif concerns the targeting cellular proteins to the proteasome for degradation. However, it also seems that this motif enables the HPV E6 protein to bind to tumour suppressor proteins. In HPV infected cells this motif therefore no longer functions as a PDZ degradation signal and, moreover, it inactivates the tumour suppressor protein. For these, non-limiting, reasons it is thought that this motif characterises high-risk viral types.
Figure 12 shows primer target sites within a range of viruses, including high-risk viruses, and also corresponding probe target sites. Moreover, there is shown reverse primer target sites for each of the relevant viruses. Advantageously, the self-probing amplicon will contain a virus primer that is specific for the region of the nucleic acid molecule encoding the aforementioned motif and, ideally, a degenerate virus probe. However, other combination of primer and probe sequences fall within the scope of the invention. For example, the self-probing amplicon may contain a degenerate virus primer and a virus probe that is specific for the nucleic acid encoding the afore motif and so specific for a high- risk virus. Alternatively, the self-probing amplicon primer and probe may both be degenerate and it is the reverse primer that is specific for a high-risk virus.
Conveniently, to avoid multiple testing of a sample using many different 'virus self-probing amplicons', there is provided a self-probing amplicon of the invention wherein the target comprises more than one nucleic acid sequence from more than one virus and/or the virus primer component exhibits some degeneracy with respect to the target, whereby the virus primer is not entirely complementary to each one of the nucleic acid sequences of the target. However, for screening purposes, it may be more convenient to carry out a preliminary assay to establish first whether there is any of a virus or category of virus present in the sample and, if this assay were to prove positive, then to proceed to the specific typing and quantitation assays enabled by the method of this invention. Such a preliminary assay may comprise any previously known method for virus detection, including those mentioned hereinbefore. Preferably, however, the preliminary assay comprises the method of the invention wherein a tailed primer is first incorporated into the viral primer extension product. The tail comprises a nucleic acid sequence capable of amplifying, eg under PCR conditions, the viral nucleic acid sequence of a plurality of viruses or virus, such as HPV, types. It is believed that this is the first time the concept of tailed primers has been applied to self-probing amplicons.
Accordingly, in a preferred method of the invention in order to test for any HPV type in a single reaction, a unique self-probing amplicon target site is introduced into the amplification (primer extension) product. By attaching a unique 'designer tail' to the prior art consensus primers (such as one capable of amplifying all 20 of the common HPV types), this aim can be achieved. A preferred tailed primer for use in the present invention is shown in Table 7 of
Example 5 [SEQ ID NO: 7]. Two suitable degenerate self-probing amplicons for use in the present invention are shown in Table 7 of Example 5 [SEQ ID NOs: 8 & 9]; these sequences also comprise preferred primer and probe components. This methodology is illustrated in Figure 6 where it can be seen that a tailed primer, wherein the tail contains the designer sequence ATG TGG AAA CAT GCA TGG, is integrated into viral DNA using two rounds of PCR amplification. Moreover, it follows that if the reverse primer is specific for a particular HPV or group of HPV's then the tailed primer will be integrated into a specific type of HPV. Subsequently, the use of a, relatively, degenerate self-probing amplicon that has a virus primer capable of hybridising to the "designer" sequence that has been incorporated into the HPV to be detected, results in the self-probing amplicon binding to the target nucleic acid followed by primer extension and the subsequent binding of the virus probe to its target nucleic acid in the virus primer extension. It will be apparent to those skilled in the art that the "designer" section of the tailed primer may be any preferred combination of nucleic acids. Thus the illustration herein is not intended to be limited.
Accordingly, the present invention further provides the novel tail part [SEQ ID NO: 10] of these sequences; the probe part of these sequences being designed to bind to the known GP6+ sequence.
SEQ ID NO: 10 ATGTGGAAACATGCATGG GP6+ tail comprised in
[SEQ ID NO:7]
The present invention also provides a diagnostic kit for use in a method of the invention, which kit comprises one or more of the virus self-probing amplicons, housekeeping or control self-probing amplicons or tailed primers of the invention. Particularly preferred is a kit suitable for use in determining viral load per cell, and accordingly comprises at least two, and suitably four or more, self- probing amplicons, including at least one housekeeping self-probing amplicon and, optionally, a tailed primer and/or a reverse primer that is specific for a particular type of HPV or group of HPV's such as high-risk HPV's or low risk HPV's. Also preferred is a kit comprising at least one self-probing amplicon for targeting at least one of the E1 , E2, E6 and E7 HPV genes. In the instance where the reverse primer is specific for a high-risk HPV, it is designed to, ideally, bind to the part of the nucleic acid that encodes the PDZ domain of the E6 protein and so is designed to bind to the target sites, for the high-risk viral types, shown in Figure 12.
Furthermore, the invention provides the use of such self-probing amplicons, including the probe and primer components thereof, in a method as per below or in the preparation of a kit as hereinbefore described.
The invention further provides a method for one or more of:
(a) detection;
(b) typing;
(c) determination of viral load per cell; and/or (d) determination of the integration state of a virus in a sample suspected of comprising one or more target viral nucleic acid sequence(s),
which method comprises: (IA) contacting the sample with a self-probing amplicon ( irus self-probing amplicon') comprising (i) a virus primer capable of hybridising to at least one target viral nucleic acid sequence and undergoing amplification thereof under primer amplification conditions to form a virus primer extension product; (ii) a virus probe comprising a nucleic acid sequence complementary to a target sequence of the virus primer extension product and capable of hybridisation thereto, provided that the self-probing amplicon is adapted to ensure that the virus probe is unresponsive to amplification under the primer amplification conditions; and (iii) a member of a virus signalling system, which system is capable of causing a detectable signal to be effected on hybridisation of the virus probe sequence to the virus primer extension product, whereby presence or absence of the target viral nucleic acid sequence in the sample is indicated by the detectable signal; and, optionally,
(IB) amplifying the product of step (IA) under the primer amplification conditions to an extent enabling the detectable signal to be effected after step (II); then
(II) separating the virus primer extension product from the target viral nucleic acid sequence; allowing the virus probe to hybridise to the target sequence of the virus primer extension product; and monitoring the signalling system, wherein the 'viral self-probing amplicon(s)' is/are adapted to allow detection, quantification or assessment of the E1 , E2, E6 and/or E7 HPV genes.
The invention further provides a method comprising the following steps:
(0)(A) contacting a target viral nucleic acid sequence from the sample with 'tailed primer', which comprises: (i) a primer region comprising a nucleic acid sequence ('consensus primer sequence') complementary to a consensus sequence of the viral nucleic acid sequence and capable of hybridisation thereto and undergoing amplification thereof under primer amplification conditions to form a tailed primer extension product; and (ii) a tail region comprising a unique sequence not present in or prepared by any component of this method ('designer' sequence); and
(0)(B) carrying out at least two rounds of amplification under the primer amplification conditions, whereby the 'designer' sequence becomes incorporated into the primer extension product; and
wherein the primer component of the 'virus self-probing amplicon' is capable of binding to the 'designer' sequence and the probe component of the virus self- probing amplicon is complementary to the consensus primer sequence. In a preferred method of the invention said virus primer of the self-probing amplicon hybridises to at least one target high-risk viral nucleic acid and so, ideally, includes a nucleic acid sequence that is capable of hybridising to nucleic acid encoding a motif, as herein described, that characterises a high-risk virus or to a primer target site shown in Figure 12. Alternatively, the virus probe of the self-probing amplicon hybridises to at least one target high-risk viral nucleic acid as afore described.
Additionally, or alternatively, in the methodology of the invention which involves primer amplification, the reverse primer is designed to hybridise to nucleic acid encoding a motif, as herein described, that characterises a high-risk virus or to the reverse primer target 3' - 5' antisense sequence shown in Table 12.
The present invention will now be illustrated by the following Examples.
EXAMPLE 1 - Viral Detection using VESPA
General Materials and Methods
Cell Lines The HeLa, Caski and SiHa cell lines were provided by Dr Steve Man, University of Wales College of Medicine, Cardiff, UK, and are available from the ATCC (American Type Culture Collection, http://www.atcc.org/) Clinical Samples
Patients were either recruited during routine colposcopy clinics at Llandough Hospital, Cardiff, UK, or as part of an MRC field study in West Africa. Informed consent was obtained from all subjects who were asked for permission to assess their smear for HPV, but the methodology remained confidential.
Cervical samples were collected using conical cytobrushes, and transported in 0.5ml of Digene transport medium (from Silver Spring, Md., USA). Samples taken in the UK were stored at 4°C for up to 24 hours before processing. Samples collected in the Gambia were stored, frozen in liquid nitrogen, and processed within one month. Biopsies were collected, with informed consent, from patients undergoing treatment for Recurrent Respiratory Papillomatosis and cervical cancer at The University Hospital of Wales, Cardiff, UK and Llandough Hospital, Penarth, UK.
DNA Purification
DNA was purified from cell lines by re-suspension of cells in 640 I of Nuclear Lysis Buffer (10mM Tris HCI, 0.4M NaCl, 2mM ethylenediamine tetra-acetate pH 8.0, 10% sodium dodecyl sulphate), 100 I of 6M NaCl and 740 I of chloroform. The solution was thoroughly mixed, centrifuged and the top phase extracted. DNA was precipitated by the addition of 1ml 95% ethanol and pelleted by centrifugation. The pellet was washed twice with 70% ethanol, dried in a rotary evaporator and re-suspended in 500 I of de-ionised water. DNA was purified from cervical brush samples by a simple modification (squeezing the cytobrushes on the side of the tube, and freezing times were increased to 24 hours from 2 hours) of the freeze-thaw method as described by Jacobs et al in Jacobs et al J Clin Microbiol 35 791-5 (1997). Epithelial cells obtained from the cytobrush samples were pelleted by centrifugation and re-suspended in 1ml 10mM Tris pH 7.4, and frozen at -70°C for 24 hours. A 100 I aliquot was thawed, boiled for 10 minutes; chilled on ice; spun at 13,000 rpm in a microfuge for 3 minutes; and supernatant decanted and stored. DNA was extracted from biopsy material by incubation in 1 ml 10mM Tris HCI pH7.4 containing 10mg/ml proteinase K (from Sigma, UK) for 1 hour at 56°C before boiling to activate the enzyme.
PCR EIA - Comparative Method
PCR-EIA was performed as described by Jacobs et al (q.v.).
VESPA Method Primer Design
Table 6 shows the sequences of the six self-probing amplicon primers used in this study. All primers were synthesised by Oswel Research Products, Southampton S016 7PX, UK.
All self-probing amplicon primers were purified by double HPLC. Standard primers were purified by gel filtration. Primers were designed to detect the HPV L1 gene. Primer locations varied between types but were positioned approximately between 6600 and 6750 bp. The forward primer sequence of each self-probing amplicon is type-specific, and is located at the same sequence position as that of the GP6+ primer described by Jacobs et al (q.v.). Self-probing amplicon probe sequences were designed by aligning the L1 open reading frames (ORF) of twenty common HPV types (HPV-6, 11 , 16, 18, 31 , 33, 39, 40, 42, 43, 44, 45, 51 , 52, 56, 58, 59, 66 and 68)
(http://hpy-web.lanl.gov). The areas of greatest sequence variation adjacent to the GP6+ primer binding site of Jacobs et al (q.v.) were selected as the probe target binding site. The probe sequence of these primers was checked against 70 common papillomavirus sequences and no significant homology was found. The SC16 primer component comprises the known GP6+ sequence. The reverse primer target sequence is the GP5+ sequence of Jacobs et al (q.v.).
Primer Validation - VESPA v Reference Cell Lines
The HPV 16 and 18 primers were tested for specificity using reference cell lines with integrated HPV DNA. The Caski cell line contains 60-600 copies of the
HPV-16 ORF per cell, and the HeLa cell line contains 10-50 copies of the HPV- 18 L1 ORF.
Figure 1a shows the results of PCR reactions using the Sc16 self-probing amplicon primer (designed for detection of HPV-16 DNA), and DNA extracted from the HPV-16 positive Caski cell line, DNA from the HPV-18 positive HeLa cell line and a negative control (no DNA). A significant increase in fluorescence was only detected with the HPV-16 containing Caski DNA. Figure 1 b shows a similar experiment using the Sc18 self-probing amplicon primer in place of Sc16. Here, significant fluorescence was only detected with the HPV-18 positive HeLa cell line. The primers were then used to detect HPV- 16 and -18 in clinical samples previously typed using PCR-EIA (Figure 2c and Figure 2d - see below).
Detection of HPV by VESPA
PCR amplification of 1 μl aliquots of DNA solution were performed using 0.5 μM
self-probing amplicon primer and 0.5 μM GP5+ reverse primer as described by
Jacobs et al (q.v.) in a total reaction volume of 10 μl. Reactions were performed
using a Light Cycler (available from Bio/Gene, Kimbolton, Cambs, PE18 ONJ or Roche Diagnostics Ltd, Bell Lane, Lewes, East Sussex BN7 1LG) and run for 100 cycles under the following cycling parameters: 96°C-1s, 40° C-5s, 72° C-1s. Reaction conditions were as follows: 200 μM dNTP's, 4mM MgCI2, 50mM Tris
HCI pH 8.9, 10mM ammonium sulphate, 0.1 % Tween™ 20, bovine serum albumin 250ng/μl, and 0.5 U/μl Taq polymerase (from Advanced
Biotechnologies, Epsom, Surrey, UK). Fluorescence was detected in channel one (530nm) at 40°C.
PCR control reactions were carried out as above but included 1 μl SYBR Gold
(from Bio/Gene, Kimbolton, Cambs, PE18 ONJ). Self-probing amplicon control reaction (negative controls) contained 1 I H20 in place of DNA. The VESPA technique was then extended to enable detection of HPV types -6, - 11 , -31 , -33, -39, -51 and -56. As there are no commercially-available cell lines containing these HPV types, primer specificity was validated using clinical samples previously tested using PCR-EIA as shown below in Table 5, with reference to Example 3. Figure 2 (a-h) shows positive results from typing reactions for HPV -6, -11 , -16, -18,-31 , -33, -39, and-51 , respectively.
EXAMPLE 2 - Viral Load Determination using VESPA
A theoretical advantage of self-probing amplicon PCR is its ability to determine viral load. Shown, in Figure 3, are the results of Sc16 typing reactions performed using a dilution series of the SiHa cell line (one to two copies of HPV- 16 per cell). The dilution series from 50,000 to 500 HPV copies per cell were clearly distinguishable, and the signal for HPV-16 remained positive in the sample containing a single copy of HPV-16 DNA.
In order to explore the possibility of establishing a technique capable of determining viral load per cell, a primer was designed to detect (human) beta- globin DNA (ScBG, SEQ ID NO: 11 in Table 3). Table 3 - HPV-16 Viral Load - Sequences of VESPA Primers.
Figure imgf000032_0001
Sc = self-probing amplicon; FAM = Fluorophore = 6-carboxyfluorescein. MR = Fluorescence quencher = Methyl Red; HEG = PCR blocker = hexethyl glycol. Sequences in bold typeface are novel probe sequences
Figure 4 shows the results of an experiment conducted using a self-probing amplicon designed to detect the human beta-globin gene over the same dilution series of SiHa cells used above for HPV-16. Once again, the signal remained positive down to a single cell and is quantitative at and above 50 copies per cell. Figure 5 shows a plot of the ratio of the fluorescence produced by Sc16 and ScBG against the logarithm of the viral copy number per cell of target DNA. A range of copy numbers per cell may be calculated from a cell line containing a fixed copy number, by calculating the ratio of two different dilutions. For example, the Sc16 Fmax value for 5000 cells can be divided by that for the ScBG Fmax for 100 cells to obtain a value for a notional cell line containing 50 copies per cell (5000/100). Disease grade was determined by cytology. VL ratio (Viral Load) was measured in RSU (Relative Self-probing amplicon Units) by dividing Sc16 Fmax by ScBG Fmax- Viral copies per cell were estimated using the standard curve shown in Figure 5.
Table 4 - Viral Load Data
Sample HPV-Type Disease VL ratio (RSU) Av.Viral Copies per cell Grade
1 2885C 16 Normal -0.3508 Undetectable
2 2503F 16 Normal -0.1204 Undetectable
3 0155H 16 Normal -0.1007 Undetectable
4 LD20 6,16,39 CIN2 -0.0242 Undetectable
5 0119D 16 Normal -0.0166 Undetectable
6 SJ 16 Ca vagina 0.0095 0.042
7 LD49 16 CIN2-3 0.0331 0.047
8 AD 16 CIN2-3 0.0550 0.052
9 LD19 16 CIN2 0.0697 0.056
10 LD15 16 CIN3 0.0750 0.057
11 LD25 16,31 CIN3 0.0786 0.058
12 LD24 16 CaCx 0.1641 0.088
13 LD50mm 16 CIN3 0.4945 0.441
14 2503F 16 CIN2-3 0.5965 0.724
15 LD45 16 CIN2-3 0.7601 1.603
16 0093A 16 CIN2-3 0.8749 2.805 Shown in Table 4 are the results of applying this viral load determination technique to 16 clinical samples previously found (by Jacobs et al, q.v.) to be HPV-16-positive using PCR-EIA. The most striking finding from these experiments is that the four cervical smears with normal cytology have low viral loads using VESPA, and the only other sample with a low viral load, but significant neoplasia, is co-infected with HPV-6 and HPV-39. The preliminary viral load data shown in Table 4 suggest that the presence of cervical neoplasia might correlate with viral load.
EXAMPLE 3 - Comparison of VESPA with PCR-EIA
In order to test the suitability of VESPA for HPV typing from cervical smears in the clinical setting, the self-probing amplicon primers were used to test 108 samples previously HPV typed (Jacobs et al q.v.), using PCR-EIA. In order directly to compare the two techniques, DNA extraction was performed using the freeze-thaw method described by Jacobs et al, q.v. This technique is sub- optimal for PCR amplification using self-probing amplicon primers. The less efficient freeze-thaw DNA extraction method was used in this study, since this method is recommended for PCR-EIA and we wished to compare PCR-EIA and VESPA using identical DNA samples. The results of these experiments are shown in Table 5.
Selected positive sample types have subsequently been confirmed by direct sequencing (data not shown). The overall concordance between VESPA and PCR-EIA is 94%, with a kappa value of 0.89 indicating good agreement. Indeed, a similar concordance figure was achieved when PCR-EIA was performed on identical samples by different reference laboratories described by Jacobs et al q.v. Of 108 samples, VESPA failed to detect five incidences of HPV-16 and one of HPV-18. There were no false positives.
There are several explanations for the small number of remaining discrepancies observed between VESPA and PCR-EIA. First, the results produced by PCR- EIA might be false positives. There is evidence from the study comparing PCR- EIA in several different laboratories that it is prone to the occasional false positive. The fact that four of the six discrepant samples have normal cytology supports this supposition. Of the remaining two, one has low-grade lesions and for the other we have no clinical data. Secondly, the samples may be positive for HPV but contain an intra-typic HPV variant containing polymorphism within the probe-binding site. Self-probing amplicon probes discriminate between sequences on the basis of a single base change, whereas PCR-EIA probes are more tolerant of sequence variation. In this regard, it may be relevant that 5/6 discordant samples were obtained from West Africa, where there is likely to be more variation within the probe binding site. Table 5 - Comparison of PCR-EIA and VESPA
Figure imgf000036_0001
Figure imgf000037_0001
/f-values were calculated as described by Armitage et al in Statistical Methods in Medical Research, Third Edition (Oxford; Blackwell Scientific Publications)
EXAMPLE 4 - VESPA to detect Integration Status - Calculation of E6/E7:E1/E2
Self-probing amplicon sequences designed for the assessment of HPV integration state are shown in Table 6. They are used in accordance with the method described in the Examples above. Integration status is measured as described hereinabove.
TABLE 6
Figure imgf000037_0002
Figure imgf000038_0001
Key: Sc = self-probing amplicon; RP = reverse primer Bold typeface indicates probe and primer sequences, respectively.
EXAMPLE 5 - VESPA using Tailed Primers for Degenerate HPV Detection
A suitable strategy comprises the following steps, illustrated by Figure 6: 1. A primer containing a 'designer tail' is used to amplify viral DNA (if present). A standard reverse primer is also included. Alternatively, a HPV specific reverse primer may be included so ensuring that the "designer tail" is integrated into a specific type of HPV and so making the following methodology specific for the detection of a specific type of HPV. Alternatively still, a HPV specific reverse primer may be engineered so that it identifies one or more HPV types and so a target group of HPV viruses may be detected. As the skilled artisan will appreciate when performing this methodology, any one or more HPV specific reverse primers may be used so as to target the nature of the HPV nucleic acid that is to be detected. 2. Amplification proceeds as in a standard reaction. 3. After the first round of amplification, the 'designer' sequence has no complementary sequence.
4. After the second round of amplification, the 'designer' sequence is incorporated and is amplified in each round.
5. In a separate reaction, this amplification can then be detected using a self-probing amplicon comprising a primer targeting the 'designer' sequence and a probe for detecting the consensus primer. It may be necessary to use two self-probing amplicons with a slight overhang into the viral DNA to avoid unacceptable background fluorescence caused by primer dimer.
Figure 11 is a schematic illustration of the methodology that is described above.
As can be seen, on the left-hand side, DNA from a cervical smear (or any other tissue that may contain HPV) is added to one of two tubes. Tube 1 contains at least one degenerate Scorpion or virus self-probing amplicon with a virus primer capable of hybridising to "designer sequence"; a tailed primer wherein the tail comprises a "designer sequence" and one or more reverse primers that are designed to hybridise to a region of a high-risk HPV, plus appropriate reagents. Tube 2 contains a degenerate Scorpion or virus self-probing amplicon with a virus primer capable of hybridising to a "designer sequence"; a tailed primer wherein the tail comprises a "designer sequence" and at least one reverse primer that is specific for at least one low-risk HPV, plus appropriate reagents. If HPV is present in the cervical smear sample then, if the HPV is a high-risk HPV, the tailed primer and the reverse primer with the high-risk specific sequence will bind thereto and amplification will take place resulting in the "designer sequence" being incorporated into the viral DNA. Thereafter, the virus self- probing amplicon will bind, via its virus primer, to the "designer sequence" that has been incorporated into the viral DNA and the virus self-probing amplicon will therefore operate in such a fashion that a primer extension is produced and the virus signalling system will be activated resulting in a signal, typically a fluorescent signal, being emitted. Thus a fluorescent signal will signify the presence of high-risk HPV in the original sample.
Exactly the same logic applies, mutatis mutandis, to Tube 2 except, in this instance, fluorescence will only take place if there is a low-risk HPV in the sample.
On the right-hand side of Figure 11 is shown the control experiments represented by Tubes 3 and 4. Tube 3 has the same contents as Tube 1 and
Tube 4 has the same contents as Tube 2. Given that water is added to Tubes 3 and 4, there should be no HPV in the water sample and therefore no fluorescent signal should be produced.
With the above schematic it can be seen that there are four possible results.
1. Where the fluorescence emitted from Tube 1 is greater than Tube 3, one would conclude that the original sample contained a high-risk HPV. 2. Where the fluorescence in Tube 2 exceeded that in Tube 4, one would conclude that the original sample contained a low-risk HPV. 3. Where the fluorescence of Tube 1 is greater than the fluorescence of Tube 3 and the fluorescence of Tube 2 is greater than the fluorescence of Tube 4. one would conclude that the original sample contained both a high-risk HPV and a low-risk HPV. 4. Where the fluorescence of Tube 1 was less than the fluorescence of Tube 3 and the fluorescence of Tube 2 was less than the fluorescence of Tube 4, one would conclude that there was no HPV in the original sample and therefore zero infection.
Using a method analogous to that described in previous Examples, the following tailed primers were used to detect degenerate HPV (Table 7):
Table 7: Tailed Primers
Figure imgf000041_0001
Key: Sc = self-probing amplicon; FAM = Fluorophore = 6-carboxyfluorescein MR = Fluorescence quencher = Methyl Red; HEG = PCR blocker = hexethyl glycol
Sequence in bold typeface is novel tail region. Both primer and probe regions are also novel.
Figure 7 demonstrates the ability of the degenerate self-probing amplicon mix to detect HPV types -6, -16 and -18. Comparisons may be performed using samples pre-amplified under conditions described by Jacobs et al (qv). Interestingly, two of these samples were barely visible after agarose gel electrophoresis with ethidium bromide staining, suggesting that the VESPA approach produces the expected improvement in sensitivity.
Assay Conditions
All amplifications were carried out using good laboratory practise, filter tips and in a laboratory not previously exposed to polyomaviruses.
First Round Amplification (Pre-amplification).
Each reaction was set up three times with appropriate primer pairs in order to test for SV40, JC and BK.
51 of each sample per reaction was pre-amplified under the following conditions; 0.5 M P1 and P3 (see Table 8), 2.5mM MgCI2, 200 M dNTPs, 1.25U of Taq polymerase (Advanced Biotechnologies, Epsom, Surrey, UK) and 5 I of 10X reaction buffer (Perkin-Elmer Gold) in a total reaction volume of 50 I. Each sample was also SV40-typed under the conditions described above and using the P1-P2 primer pair (Table 8).
Second Round Amplification (Amplicon Detection).
Assay set-up was performed in a laboratory physically separated (100m) from that used for the first round amplification.
Amplicon detection was performed in two different real-time PCR machines: (i) LightCycler (Roche Diagnostics Ltd, Bell Lane, Lewes, East
Sussex BN7 1LG) and
(ii) iCycler (Bio-Rad Laboratories Ltd., Bio-Rad House, Maylands
Avenue, Hemel Hempstead, Herts. HP2 7TD); and under slightly different conditions.
PCR amplification of 1 I aliquots of DNA (2 I iCycler) solution were performed using 0.5 M Scorpion primer (see Table 1) and 0.5 M reverse primer in a total reaction volume of 10 I (20 I iCycler). Reaction conditions were as follows: 200 M dNTPs, 4mM MgCI2, 50mM Tris HCI pH 8.9, 10mM ammonium sulphate, 0.1 % Tween 20, bovine serum albumin 250ng/ I, and 0.5 U/ I Taq polymerase (Advanced Biotechnologies, Epsom, Surrey, UK). Fluorescence was detected in channel one/530nm (FilterSet4 iCycler) and at 40°C (45°C iCycler). Self-probing amplicon control reaction (negative controls) contained H20 in place of DNA. All primers were synthesised by Oswel Research Products, Southampton S016 7PX, UK. Cycling Parameters Pre-amplification 94°C for 180s followed by 30 cycles of 94°C-60s, 52°C-60s and 72°C-60s and finally 72°C-300s.
Amplicon Detection i) LightCycler. 100 cycles of 96°C-1 s, 40° C-5s, 72° C-1 s. (ii) iCycler. 95°C for 180s followed by 40 cycles of 95°C-30s, 45°C-30s,
72°C-15s.
Example 7 - VESPA in HPV Associated with RRP
Clinical Samples
Patients were recruited by contacting all Ear, Nose and Throat surgeons throughout Wales and all those in England known to be actively engaged in recurrent respiratory papillomatosis (RRP) research. All cases of RRP, as confirmed by characteristic histological changes on biopsy and/or those requiring more than 2 surgical interventions, were identified. Informed consent was obtained from all subjects. Biopsies were collected, with informed consent, from patients undergoing treatment for Recurrent Respiratory Papillomatosis at the University Hospitals of Wales, Cardiff and Sheffield. Disease Severity
The grade of clinical disease was assigned using the parameters outlined by Derkay et al in Laryngoscope 108(6) 935-7 (1998); this equation accounts for both the number and time interval between surgeries.
DNA Purification
DNA was extracted from biopsies by incubating approx 1mm3 of biopsy material in 1ml 10mM Tris hydrochloride pH7.4 containing 0.5mg/ml proteinase K (Sigma, UK) for 1 hour at 56°C. 100 μl aliquots were then taken and boiled for 10 minutes, cooled on ice for 5 minutes, spun at 13,000 rpm for 3 minutes (in order to remove particulate matter) and decanted into fresh tubes.
PCR EIA
In order to confirm HPV types, the reference technique of Jacobs et al (qv) for PCR-EIA was performed on all samples as described in Example 3.
Results
The viral loads presented in Table 10 were calculated as described in Example 2. Briefly, the ratio of the fluorescence signals produced using a cognate HPV- self-probing amplicon and that of a self-probing amplicon designed to detect a human genomic beta-globin housekeeping gene was calculated.
Although there are clearly examples of patients with high viral load and mild disease (NL) and wee versa (DN), a patient who is in remission had the lowest viral load and the most severe patient had the highest viral load. This study may suggest that the most significant prognostic marker in RRP is HPV-type; HPV-6 leads to mild and HPV-11 to aggressive disease.
Table 10
Figure imgf000046_0001
Organization Applicant Street : 30-36 Newport Road City : Cardiff State : Country : United Kingdom PostalCode : CF24 ODE PhoneNumber : FaxNumber : EmailAddress : <110> OrganizationName : UNIVERSITY COLLEGE CARDIFF CONSULTANTS LTD
Individual Applicant Street : Commercial Development Group, RACD, 7th Floor, 30-36 Newport Road, City : Cardiff State : Country : United Kingdom PostalCode : CF24 ODE PhoneNumber : FaxNumber : EmailAddress : <110> LastName : HART <110> FirstName : Keith <110> Middlelnitial : <110> Suffix :
Application Project
<120> Title : Virus Detection Method, Primers Therefor and Screening Kit <130> AppFileReference : WCM.82D <140> CurrentAppNumber : GB 0425085.8 <141 > CurrentFilingDate : 2004-11-13
Earlier Applications
<150> PriorAppNumber : GB 0423138.7 <151> PriorFilingDate : 2004-10-19
Earlier Applications
<150> PriorAppNumber : GB 0410892.4 <151 > PriorFilingDate : 2004-05-15
Earlier Applications
<150> PriorAppNumber : GB 0408409.1 <151 > PriorFilingDate : 2004-04-15
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : ccgcggcaaa tgtacaatca ttaccgcgg 29
<212> Type : DNA
<211 > Length : 29 SequenceName : WCM.82D - SEQ ID 1 SequenceDescription :
Page 1 Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cagaatggat acaaagacaa acagt 25
<212> Type : DNA
<211> Length : 25 SequenceName : WCM.82D - SEQ ID 2 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cccgcggacg taggtctgta ctatcatttt catccgcggg 40
<212> Type : DNA
<211> Length : 40 SequenceName : WCM.82D - SEQ ID 3 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : ccaacgttta aatgtgtgtc agga 24
<212> Type : DNA
<211 > Length : 24 SequenceName : WCM.82D - SEQ ID 4 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : ccgccacatt ctaatattat atcatgtata ggcgg 35
<212> Type : DNA
<211> Length : 35 SequenceName : WCM.82D - SEQ ID 5 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : aagttaccac agttatgcac agagc 25
<212> Type : DNA
<211> Length : 25 SequenceName : WCM.82D - SEQ ID 6 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgtggaaac atgcatggcg acatgaaaaa taaactgtaa atcatatct 49
<212> Type : DNA
<211> Length : 49 SequenceName : WCM.82D - SEQ ID 7 SequenceDescription :
Paqe 2 Sequence
<213> OrganismName : Virus
<400> PreSequenceString : ccgcgggaag aatatgattt acaccgcggg tggaaacatg catggcgac 49
<212> Type : DNA
<211> Length : 49 SequenceName : WCM.82D - SEQ ID 8 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : ccgcgggagg aatatgattt acaccgcggg tggaaacatg catggcgac 49
<212> Type : DNA
<211> Length : 49 SequenceName : WCM.82D - SEQ ID 9 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgtggaaac atgcatgg 18
<212> Type : DNA
<211> Length : 18 SequenceName : WCM.82D - SEQ ID 10 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : ccgcggatgg tgtctgtttg agccgcggac acaactgtgt tcactagc 48
<212> Type : DNA
<211> Length : 48 SequenceName : WCM.82D - SEQ ID 11 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : ccgcggcaaa tgtacaatca ttaccgcggc agaatggata caaagacaaa cagt 54
<212> Type : DNA <211> Length : 54 SequenceName : WCM.82D - SEQ ID 12 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cccgcggacg taggtctgta ctatcatttt catccgcggg ccaacgttta aatgtgtgtc 60 agga 64
<212> Type : DNA
Pacte 3 <211> Length : 64 SequenceName : WCM.82D - SEQ ID 13 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : ccgccacatt ctaatattat atcatgtata ggcggaagtt accacagtta tgcacagagc 60
<212> Type : DNA <211> Length : 60 SequenceName : WCM.82D - SEQ ID 14 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cacgtagaga aacccagctg 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 15 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : gacgcagaga aacacaagta 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 16 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : ctcgtactga aacccaagtg 20
<212> Type : DNA
<211 > Length : 20 SequenceName : WCM.82D - SEQ ID 17 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : gacgtagaga aactgcactg 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 18 SequenceDescription :
Sequence
<213> OrganismName : Virus <400> PreSequenceString :
Page 4 cacgtagaga aaccgaggtg 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 19 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cacgaagaga aacccaagta 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 20 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : gacgtaggga aacacaagta 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 21 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : aacgtaacga aacccaagtg 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 22 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : gccgacctgt gacccaagtg 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 23 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : aacctagaga atctacagta 20
<212> Type : DNA
<211 > Length : 20 SequenceName : WCM.82D - SEQ ID 24 SequenceDescription :
Sequence
<213> OrganismName : Virus
Page 5 <400> PreSequenceString : gacgtagaca aacacaagtg 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 25 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cgcgtagtga aacactggtg 20
<212> Type : DNA
<211> Length : 20 SequenceName : WC .82D - SEQ ID 26 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : aagctacaga atctacagta 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 27 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : gcgtggaaga catgttaccc 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 28 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : gcgtggaaga cttgttaccc 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 29 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : aacgctcgga gacattgtgt 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 30 SequenceDescription :
Sequence
Paαe 6 <213> OrganismName : Virus
<400> PreSequenceString : gcgtggagag acgcctaccc 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 31 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : gacgatcaga gactatgtgc 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 32 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : gcgtggaaac tatactacct 20
<212> Type : DNA
<211> Length : 20 SequenceName : WCM.82D - SEQ ID 33 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgcatgga
<212> Type: DNA
<211 > Length : 9 SequenceName : WCM.82D - SEQ ID 34 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgcatgga
<212> Type : DNA
<211> Length : 9 SequenceName : WC .82D - SEQ ID 35 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgcgtgga
<212> Type : DNA
<211 > Length : 9 SequenceName : WCM.82D - SEQ ID 36 SequenceDescription :
Sequence
Paqe 7 <213> OrganismName : Virus <400> PreSequenceString : atgagagga <212> Type : DNA <211 > Length : 9 SequenceName : WCM.82D - SEQ ID 37 SequenceDescription :
Sequence
<213> OrganismName : Virus <400> PreSequenceString : atgcatgga <212> Type : DNA <211> Length : 9 SequenceName : WCM.82D - SEQ ID 38 SequenceDescription :
Sequence
<213> OrganismName : Virus <400> PreSequenceString : atgcgtgga <212> Type : DNA <211> Length : 9 SequenceName : WCM.82D - SEQ ID 39 SequenceDescription :
Sequence
<213> OrganismName : Virus <400> PreSequenceString : atgcatgga <212> Type : DNA <211> Length : 9 SequenceName : WCM.82D - SEQ ID 40 SequenceDescription :
Sequence
<213> OrganismName : Virus <400> PreSequenceString : atgcgtggt <212> Type : DNA <211> Length : 9 SequenceName : WCM.82D - SEQ ID 41 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgcgtgga
<212> Type : DNA
<211> Length : 9 SequenceName : WCM.82D - SEQ ID 42 SequenceDescription :
Paqe 8 Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgcatggt
<212> Type : DNA
<211> Length : 9 SequenceName : WCM.82D - SEQ ID 43 SequenceDescription :
Sequence
<213> OrganismName : Virus <400> PreSequenceString : atgagagga <212> Type : DNA <211> Length : 9 SequenceName : WC .82D - SEQ ID 44 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgcatgga
<212> Type : DNA
<211> Length : 9 SequenceName : WCM.82D - SEQ ID 45 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgcatggt
<212> Type : DNA
<211> Length : 9 SequenceName : WCM.82D - SEQ ID 46 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgcatgga
<212> Type : DNA
<211> Length : 9 SequenceName : WCM.82D - SEQ ID 47 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgcatgga
<212> Type : DNA
<211> Length : 9 SequenceName : WCM.82D - SEQ ID 48 SequenceDescription :
Page 9 Sequence
<213> OrganismName : Virus <400> PreSequenceString : atgcatgga <212> Type : DNA <211> Length : 9 SequenceName : WCM.82D - SEQ ID 49 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : atgcgtgga
<212> Type : DNA
<211> Length : 9 SequenceName : WCM.82D - SEQ ID 50 SequenceDescription :
Sequence
<213> OrganismName : Virus <400> PreSequenceString : atgcatgga <212> Type : DNA <211> Length : 9 SequenceName : WCM.82D - SEQ ID 51 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgacctacat tgctatgagc aatt 24
<212> Type : DNA
<211> Length : 24 SequenceName : WCM.82D - SEQ ID 52 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgatctctac tgttatgagc aatt 24
<212> Type : DNA
<211> Length : 24 SequenceName : WCM.82D - SEQ ID 53 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgaccttcta tgtcacgagc aatt 24
<212> Type : DNA
<211> Length : 24 SequenceName : WCM.82D - SEQ ID 54
Paαe 10 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgacctccac tgttatgagc aatt 24
<212> Type : DNA
<211 > Length : 24 SequenceName : WC .82D - SEQ ID 55 SequenceDescription :
Sequence
<213 OrganismName : Virus
<400> PreSequenceString : tgacctatac tgctatgagc aatt 24
<212> Type : DNA
<211> Length : 24 SequenceName : WCM.82D - SEQ ID 56 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgacctatac tgttatgagc aatt 24
<212> Type : DNA
<211> Length : 24 SequenceName : WCM.82D - SEQ ID 57 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgaccttgta tgtcacgagc aatt 24
<212> Type : DNA
<211 > Length : 24 SequenceName : WCM.82D - SEQ ID 58 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgacctgttg tgttacgagc aatt 24
<212> Type : DNA
<211> Length : 24 SequenceName : WCM.82D - SEQ ID 59 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgacttgcaa tgctacgagc aatt 24
<212> Type : DNA
<211> Length : 24
Page 11 SequenceName : WCM.82D - SEQ ID 60 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgacctacac tgctatgagc aatt 24
<212> Type : DNA
<211> Length : 24 SequenceName : WCM.82D - SEQ ID 61 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgacctacag tgcaatgagc aatt 24
<212> Type : DNA
<211> Length : 24 SequenceName : WCM.82D - SEQ ID 62 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgacctattc tgctatgagc aatt 24
<212> Type : DNA
<211 > Length : 24 SequenceName : WCM.82D - SEQ ID 63 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgaccttgtg tgctacgagc aatt 24
<212> Type : DNA
<211> Length : 24 SequenceName : WCM.82D - SEQ ID 64 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : tgacctacaa tgcaatgagc aatt 24
<212> Type : DNA
<211 > Length : 24 SequenceName : WCM.82D - SEQ ID 65 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : ccagttgacc tacattgcta tgagcaatt 29
<212> Type : DNA
Page 12 <211> Length : 29 SequenceName : WCM.82D - SEQ ID 66 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cctgtagggt tacattgcta tgagcaatt 29
<212> Type : DNA
<211> Length : 29 SequenceName : WCM.82D - SEQ ID 67 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cctgtagggt tacattgcta tgagcaatt 29
<212> Type : DNA
<211 > Length : 29 SequenceName : WCM.82D - SEQ ID 68 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cctgtatgtc taaactgcaa cgagcaatt 29
<212> Type : DNA
<211> Length : 29 SequenceName : WCM.82D - SEQ ID 69 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cccattgacc tgtattgcta tgaacaatt 29
<212> Type : DNA
<211> Length : 29 SequenceName : WCM.82D - SEQ ID 70 SequenceDescription :
Sequence
<213> OrganismName : Virus
<400> PreSequenceString : cctgtaggcc tacattgcaa tgagcaatt 29
<212> Type : DNA
<211> Length : 29 SequenceName : WCM.82D - SEQ ID 71 SequenceDescription :
Page 13 SEQUENCE LISTING
<110> UNIVERSITY COLLEGE CARDIFF CONSULTANTS LTD HART, Keith
<120> Virus Detection Method, Primers Therefor and Screening Kit
<130> WCM.82D
<140> GB 0425085.8 <141> 2004-11-13
<150> GB 0423138.7 <151> 2004-10-19
<150> GB 0410892.4 <151> 2004-05-15
<150> GB 0408409.1 <151> 2004-04-15
<160> 71
<170> Patentln version 3.1
<210> 1 <211 > 29 <212> DNA <213> Virus
<400> 1 ccgcggcaaa tgtacaatca ttaccgcgg 29
<210> 2 <211> 25 <212> DNA <213> Virus
<400> 2 cagaatggat acaaagacaa acagt 25
<210> 3 <211> 40 <212> DNA <213> Virus
<400> 3 cccgcggacg taggtctgta ctatcatttt catccgcggg 40
<210> 4 <211> 24 <212> DNA <213> Virus
<400> 4 ccaacgttta aatgtgtgtc agga 24
Page 1 <210> 5 <211> 35 <212> DNA <213> Virus
<400> 5 ccgccacatt ctaatattat atcatgtata ggcgg 35
<210> 6 <211> 25 <212> DNA <213> Virus
<400> 6 aagttaccac agttatgcac agagc 25
<210> 7 <211> 49 <212> DNA <213> Virus
<400> 7 atgtggaaac atgcatggcg acatgaaaaa taaactgtaa atcatatct 49
<210> 8 <211> 49 <212> DNA <213> Virus
<400> 8 ccgcgggaag aatatgattt acaccgcggg tggaaacatg catggcgac 49
<210> 9 <211> 49 <212> DNA <213> Virus
<400> 9 ccgcgggagg aatatgattt acaccgcggg tggaaacatg catggcgac 49
<210> 10 <211> 18 <212> DNA <213> Virus
<400> 10 atgtggaaac atgcatgg 18
<210> 11 <211> 48 <212> DNA
Page 2 <213> Virus
<400> 11 ccgcggatgg tgtctgtttg agccgcggac acaactgtgt tcactagc 48
<210> 12 <211> 54 <212> DNA <213> Virus
<400> 12 ccgcggcaaa tgtacaatca ttaccgcggc agaatggata caaagacaaa cagt 54
<210> 13 <211> 64 <212> DNA <213> Virus
<400> 13 cccgcggacg taggtctgta ctatcatttt catccgcggg ccaacgttta aatgtgtgtc 60 agga 64
<210> 14 <211> 60 <212> DNA <213> Virus
<400> 14 ccgccacatt ctaatattat atcatgtata ggcggaagtt accacagtta tgcacagagc 60
<210> 15 <211 > 20 <212> DNA <213> Virus
<400> 15 cacgtagaga aacccagctg 20
<210> 16 <211> 20 <212> DNA <213> Virus
<400> 16 gacgcagaga aacacaagta 20
<210> 17 <211> 20 <212> DNA <213> Virus
<400> 17
Page 3 ctcgtactga aacccaagtg 20
<210> 18 <211 > 20 <212> DNA <213> Virus
<400> 18 gacgtagaga aactgcactg 20
<210> 19 <211> 20 <212> DNA <213> Virus
<400> 19 cacgtagaga aaccgaggtg 20
<210> 20 <211> 20 <212> DNA <213> Virus
<400> 20 cacgaagaga aacccaagta 20
<210> 21 <211> 20 <212> DNA <213> Virus
<400> 21 gacgtaggga aacacaagta 20
<210> 22 <211> 20 <212> DNA <213> Virus
<400> 22 aacgtaacga aacccaagtg 20
<210> 23 <211 > 20 <212> DNA <213> Virus
<400> 23 gccgacctgt gacccaagtg 20
<210> 24 <211 > 20
Page 4 <212> DNA <213> Virus
<400> 24 aacctagaga atctacagta 20
<210> 25 <211> 20 <212> DNA <213> Virus
<400> 25 gacgtagaca aacacaagtg 20
<210> 26 <211> 20 <212> DNA <213> Virus
<400> 26 cgcgtagtga aacactggtg 20
<210> 27 <211> 20 <212> DNA <213> Virus
<400> 27 aagctacaga atctacagta 20
<210> 28 <211> 20 <212> DNA <213> Virus
<400> 28 gcgtggaaga catgttaccc 20
<210> 29 <211 > 20 <212> DNA <213> Virus
<400> 29 gcgtggaaga cttgttaccc 20
<210> 30 <21 > 20 <212> DNA <213> Virus
<400> 30 aacgctcgga gacattgtgt 20
Page 5 <210> 31 <211> 20 <212> DNA <213> Virus
<400> 31 gcgtggagag acgcctaccc 20
<210> 32 <211 > 20 <212> DNA <213> Virus
<400> 32 gacgatcaga gactatgtgc 20
<210> 33 <211 > 20 <212> DNA <213> Virus
<400> 33 gcgtggaaac tatactacct 20
<210> 34 <211> 9 <212> DNA <213> Virus
<400> 34 atgcatgga
<210> 35 <211> 9 <212> DNA <213> Virus
<400> 35 atgcatgga
<210> 36 <211> 9 <212> DNA <213> Virus
<400> 36 atgcgtgga
<210> 37 <211> 9 <212> DNA
Paαe 6 <213> Virus
<400> 37 atgagagga
<210> 38 <211> 9 <212> DNA <213> Virus
<400> 38 atgcatgga
<210> 39 <211> 9 <212> DNA <213> Virus
<400> 39 atgcgtgga
<210> 40 <211> 9 <212> DNA <213> Virus
<400> 40 atgcatgga
<210> 41 <211> 9 <212> DNA <213> Virus
<400> 41 atgcgtggt
<210> 42 <211> 9 <212> DNA <213> Virus
<400> 42 atgcgtgga
<210> 43 <211> 9 <212> DNA <213> Virus
<400> 43 atgcatggt
Paσe 7 <210> 44 <211> 9 <212> DNA <213> Virus
<400> 44 atgagagga
<210> 45 <211 > 9 <212> DNA <213> Virus
<400> 45 atgcatgga
<210> 46 <211 > 9 <212> DNA <213> Virus
<400> 46 atgcatggt
<210> 47 <211 > 9 <212> DNA <213> Virus
<400> 47 atgcatgga
<210> 48 <211> 9 <212> DNA <213> Virus
<400> 48 atgcatgga
<210> 49 <211> 9 <212> DNA <213> Virus <400> 49 atgcatgga
<210> 50 <211> 9 <212> DNA <213> Virus
Page 8 <400> 50 atgcgtgga
<210> 51 <211> 9 <212> DNA <213> Virus
<400> 51 atgcatgga
<210> 52 <211> 24 <212> DNA <213> Virus
<400> 52 tgacctacat tgctatgagc aatt 24
<210> 53 <211> 24 <212> DNA <213> Virus
<400> 53 tgatctctac tgttatgagc aatt 24
<210> 54 <211> 24 <212> DNA <213> Virus
<400> 54 tgaccttcta tgtcacgagc aatt 24
<210> 55 <211 > 24 <212> DNA <213> Virus
<400> 55 tgacctccac tgttatgagc aatt 24
<210> 56 <211> 24 <212> DNA <213> Virus
<400> 56 tgacctatac tgctatgagc aatt 24
Page 9 <210> 57 <211> 24 <212> DNA <213> Virus
<400> 57 tgacctatac tgttatgagc aatt 24
<210> 58 <211> 24 <212> DNA <213> Virus
<400> 58 tgaccttgta tgtcacgagc aatt 24
<210> 59 <211 > 24 <212> DNA <213> Virus
<400> 59 tgacctgttg tgttacgagc aatt 24
<210> 60 <211 > 24 <212> DNA <213> Virus
<400> 60 tgacttgcaa tgctacgagc aatt 24
<210> 61 <211 > 24 <212> DNA <213> Virus
<400> 61 tgacctacac tgctatgagc aatt 24
<210> 62 <211> 24 <212 DNA <213> Virus
<400> 62 tgacctacag tgcaatgagc aatt 24
<210> 63 <211> 24 <212> DNA <213> Virus
Paαe 10 <400> 63 tgacctattc tgctatgagc aatt 24
<210> 64 <211> 24 <212> DNA <213> Virus
<400> 64 tgaccttgtg tgctacgagc aatt 24
<210> 65 <211> 24 <212> DNA <213> Virus
<400> 65 tgacctacaa tgcaatgagc aatt 24
<210> 66 <211> 29 <212> DNA <213> Virus
<400> 66 ccagttgacc tacattgcta tgagcaatt 29
<210> 67 <211> 29 <212> DNA <213> Virus
<400> 67 cctgtagggt tacattgcta tgagcaatt 29
<210> 68 <211> 29 <212> DNA <213> Virus
<400> 68 cctgtagggt tacattgcta tgagcaatt 29
<210> 69 <211> 29 <212> DNA <213> Virus
<400> 69 cctgtatgtc taaactgcaa cgagcaatt 29
<210> 70
Page 11 <211> 29 <212> DNA <213> Virus
<400> 70 cccattgacc tgtattgcta tgaacaatt 29
<210> 71 <211> 29 <212> DNA <213> Virus
<400> 71 cctgtaggcc tacattgcaa tgagcaatt 29
Pacte 12

Claims

1. A virus self-probing amplicon comprising: (i) a virus primer capable of hybridising to at least one target viral nucleic acid sequence and undergoing amplification thereof under primer amplification conditions to form a virus primer extension product; (ii) a virus probe comprising a nucleic acid sequence complementary to a target sequence of the virus primer extension product and capable of hybridisation thereto, provided that the self-probing amplicon is adapted to ensure that the virus probe is unresponsive to amplification under the primer amplification conditions; and (iii) a member of a virus signalling system, which system is capable of causing a detectable signal to be effected on hybridisation of the virus probe sequence to the virus primer extension product, whereby presence or absence of the target viral nucleic acid sequence in the sample is indicated by the detectable signal.
2. The virus self-probing amplicon according to claim 1 wherein the virus is HPV.
3. The virus self-probing amplicon according to claims 1 or 2 wherein the virus is selected from one or more of the following HPV types: 6, 11 , 16, 18, 31 , 33, 39, 40, 42, 43, 44, 45, 51 , 52, 56, 58, 59, 66 and 68.
4. A virus self-probing amplicon according to claims 1-3 wherein said amplicon is specific for HPV-16 and the virus primer comprises any one or more of the sequences selected from SEQ ID NOS: 1 , 3 or 5.
5. A virus self-probing amplicon according to claims 1-3 wherein said amplicon is specific for HPV-16 and the virus probe comprises any one or more of the sequences selected from SEQ ID NOS: 2, 4 or 6.
6. A virus self-probing amplicon according to claims 4 or 5 wherein the virus primer comprises SEQ ID NO: 1 and the virus probe comprises SEQ ID NO: 2.
7. A virus self-probing amplicon according to claims 4 or 5 wherein the virus primer comprises SEQ ID NO: 3 and the virus probe comprises SEQ ID NO: 4.
8. A virus self-probing amplicon according to claims 4 or 5 wherein the virus primer comprises SEQ ID NO: 5 and the virus probe comprises SEQ ID NO: 6.
9. A virus self-probing amplicon according to claims 1-3 wherein the virus primer comprises a sequence that hybridises to a high-risk virus.
10. A virus self-probing amplicon according to claims 1-3 wherein the virus primer comprises a sequence capable of binding to the virus primer target site shown in Figure 12.
11. A virus self-probing amplicon according to claims 1-3 wherein the virus probe comprises a sequence that hybridises to a high-risk virus.
12. A virus self-probing amplicon according to claims 1-3 wherein the virus probe comprises a sequence that hybridises to a probe target site shown in Figure 12.
13. A virus self-probing amplicon according to any preceding claim wherein the signalling mechanism is a light emitting mechanism.
14. A virus self-probing amplicon according to claim 13 wherein the light emitting mechanism comprises a chemiluminescent molecule.
15. A virus self-probing amplicon according to claim 14 wherein there is further provided a quencher molecule corresponding to said chemiluminescent molecule.
16. A method for one or more of: (a) detection; (b) typing;
(c) determination of viral lode per cell; and/or
(d) determination of the integration state of a virus in the sample suspected of comprising one or more viral target nucleic acid molecules which method comprises: (IA) contacting the sample with a self-probing amplicon ('virus self-probing amplicon') comprising (i) a virus primer capable of hybridising to at least one target viral nucleic acid sequence and undergoing amplification thereof under primer amplification conditions to form a virus primer extension product; (ii) a virus probe comprising a nucleic acid sequence complementary to a target sequence of the virus primer extension product and capable of hybridisation thereto, provided that the self-probing amplicon is adapted to ensure that the virus probe is unresponsive to amplification under the primer amplification conditions; and (iii) a member of a virus signalling system, which system is capable of causing a detectable signal to be effected on hybridisation of the virus probe sequence to the virus primer extension product, whereby presence or absence of the target viral nucleic acid sequence in the sample is indicated by the detectable signal; and, optionally,
(IB) amplifying the product of step (IA) under the primer amplification conditions to an extent enabling the detectable signal to be effected after step
(ii); then
(II) separating the virus primer extension product from the target viral nucleic acid sequence; allowing the virus probe to hybridise to the target sequence of the virus primer extension product; and monitoring the signalling system.
17. A method according to claim 16 wherein step 1A is preceded by the following:
(0)(A) contacting a target viral nucleic acid sequence from the sample with 'tailed primer', which comprises: (i) a primer region comprising a nucleic acid sequence ('consensus primer sequence') complementary to a consensus sequence of the viral nucleic acid sequence and capable of hybridisation thereto and undergoing amplification thereof under primer amplification conditions to form a tailed primer extension product; and (ii) a tail region comprising a unique sequence not present in or prepared by any component of this method ('designer' sequence); and
(0)(B) carrying out at least two rounds of amplification under the primer amplification conditions, whereby the 'designer' sequence becomes incorporated into the primer extension product; and wherein the primer component of the 'virus self-probing amplicon' is capable of binding to the 'designer' sequence and the probe component of the virus self- probing amplicon is complementary to the consensus primer sequence.
18. A method according to claim 17 wherein the "designer sequence" comprises SEQ ID NO: 10.
19. A method according to claims 17 or 18 wherein the tailed primer comprises SEQ ID NO: 7.
20. A method according to claims 16-19 wherein the virus primer capable of hybridising to at least one target viral nucleic acid sequence hybridises to a target nucleic acid sequence of a high-risk virus.
21. A method according to claim 20 wherein the virus primer hybridises to at least one high-risk primer target viral nucleic acid sequence shown in Figure 12.
22. A method according to claims 16-19 wherein the virus probe capable of hybridising to at least one target viral nucleic acid sequence hybridises to a target nucleic acid sequence of a high-risk virus.
23. A method according to claim 22 wherein the virus probe hybridises to at least one high-risk probe target viral nucleic acid sequence shown in Figure 12.
24. A method according to claims 16-23 wherein, under primer amplification conditions, a reverse primer is used and is designed to target a high-risk virus.
25. A method according to claim 24 wherein said reverse primer is designed to hybridise to a reverse primer target nucleic acid sequence shown in Figure
12.
PCT/GB2005/001366 2004-04-15 2005-04-08 Virus detection method, primers therefor and screening kit Ceased WO2005100610A2 (en)

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GB0408409.1 2004-04-15
GB0408409A GB0408409D0 (en) 2004-04-15 2004-04-15 Virus detection method, primers therefor and screening kit
GB0410892.4 2004-05-15
GB0410892A GB0410892D0 (en) 2004-05-15 2004-05-15 Virus detection method, primers therefor and screening kit
GB0423138.7 2004-10-19
GB0423138A GB0423138D0 (en) 2004-10-19 2004-10-19 Virus detection method,primers therefor and screening kit
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GB0425085.8 2004-11-13

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CN104830869A (en) * 2015-04-30 2015-08-12 珠海雅马生物工程有限公司 HPV-integrated gene sites related to occurrence of cervical carcinoma and application thereof

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ES2181765T3 (en) * 1994-02-21 2003-03-01 Stichting Res Fonds Pathologie DETECTION OF HUMAN VIRUS PAPILOMA IN A NUCLEIC ACID AMPLIFICATION PROCESS USING GENERAL PRIMERS.
EP0972013A4 (en) * 1997-03-24 2002-07-10 Res Corp Technologies Inc CELL-SPECIFIC PROMOTERS
US20060057561A1 (en) * 2001-06-14 2006-03-16 Hart Keith W Virus detection method, primers therefor and screening kit

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104830869A (en) * 2015-04-30 2015-08-12 珠海雅马生物工程有限公司 HPV-integrated gene sites related to occurrence of cervical carcinoma and application thereof

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