WO1994026934A2 - Methode de detection du papillomavirus chez l'homme - Google Patents

Methode de detection du papillomavirus chez l'homme Download PDF

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Publication number
WO1994026934A2
WO1994026934A2 PCT/US1994/005085 US9405085W WO9426934A2 WO 1994026934 A2 WO1994026934 A2 WO 1994026934A2 US 9405085 W US9405085 W US 9405085W WO 9426934 A2 WO9426934 A2 WO 9426934A2
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hpv
assay
sequence
capture
primer
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PCT/US1994/005085
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WO1994026934A3 (fr
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Janice T. Brown
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Baxter Diagnostics Inc.
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Priority to EP94917931A priority Critical patent/EP0655091A1/fr
Priority to JP6525595A priority patent/JPH07508891A/ja
Priority to AU69456/94A priority patent/AU6945694A/en
Priority to CA002139623A priority patent/CA2139623A1/fr
Publication of WO1994026934A2 publication Critical patent/WO1994026934A2/fr
Publication of WO1994026934A3 publication Critical patent/WO1994026934A3/fr

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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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma

Definitions

  • HPVs Human papillomaviruses
  • HPVs Human papillomaviruses
  • Many of these lesions are benign such as those associated with HPV 6 and HPV 11, and are considered causative of such conditions as warts, and condylomas (see Gissman, Cane. Surv., 3: 161 (1984)).
  • epidemiological and molecular studies implicate several high risk types that infect the genital tract associated with dysplasia and sometimes progress to cervical cancer (see, for example, Durst, et al., PNAS, 80: 3812 (1983)).
  • HPV 16 and HPV 18 are predominately HPV 16 and HPV 18, with HPV 31, HPV 33, and HPV 35 being of lesser significance. More recently, another HPV type associated with malignancy, HPV 44, has been identified (Lorincz, U.S. Patent No. 4,849,331).
  • HPV of any type is generally found in extremely low numbers in biological specimens. Therefore, molecular techniques must be performed for amplifying nucleic acid viral markers from very low copy number in a specimen to detectable levels.
  • Polymerase chain reaction (PCR) has been utilized to amplify HPV viral DNA in this manner, as disclosed in WO 90/02821, and Shibata, et al., J. Exp. Med., 167: 225 (DATE).
  • Other applications of PCR to HPV diagnostics are Maitland, et al., May 1988. Seventh International Papillomavirus Workshop, Abstract, p. 5 and Campione-Piccardo, et al., May 1988, Seventh International Papillomavirus Workshop.
  • nucleic acid-based assays utilize the well-known sandwich configuration in a heterogeneous format.
  • a capture oligonucleotide is chemically conjugated to a solid support such as a microtiter well or bead, the sample is added, and the target nucleic acid having base homology to capture oligonucleotide is allowed to hybridize.
  • a detection oligonucleotide hybridizes, and after a second wash to remove unhybridized detection oligonucleotide, the amount of tracer or reporter is measured, or the signal generating means produces a signal.
  • the basic problem with such sandwich assays is relatively low capture efficiency on the solid support, which may profoundly reduce sensitivity of the assay.
  • it is essential to first amplify to detectable levels only the messenger RNA (mRNA) expressed from oncogene regions (genes E6/E7) of HPV types implicated in malignant or pre-malignant cervical lesions. This not only restricts detection to malignant and pre-malignant HPV types, but also distinguishes actual oncogene expression from mere passenger presence of virus.
  • the invention contemplates a kit comprising these reagents, buffers, sample preparation solutions, solid supports, and reaction vessels.
  • a patient specimen suspected of containing messenger RNA encoded by at least one type of HPV associated with cervical dysplasia, malignant cells, or pre-malignant cells is
  • nucleic acid amplification by self sustained sequence replication utilizing two primers separated by at least ten nucleotides, at least one such primer containing a transcriptional promoter, annealing the first such primer to its complementary sequence on the target region messenger RNA, extending the 3' end of the primer by action of a strand-extending polymerase in the presence of cofactors and nucleotide triphosphates, digesting the RNA strand of the nascent RNA /DNA duplex with an enzyme having exogenous or endogenous RNAse H activity, annealing the second such primer to its complementary sequence on the resultant single stranded cDNA, primer extending the 3' end of the primer by action of a strand-extending polymerase, transcribing the double stranded DNA with a transcriptase in the presence of nucleoside triphosphates, and repeating the amplification utilizing the newly synthesized transcripts as new targets,
  • the present invention is also directed to certain primer families and selected probes for use in the HPV detection assay, and to kits for conveniently providing reagents to users.
  • Figure 1 HPV 16 genome organization. Transcription proceeds clockwise from the P 7 promotor. AE and AL are the polyadenylation sites for the early and late transcripts.
  • Figure 2 Sequence of HPV 16. The primers are indicated by underlines. Boxes indicate splice donor and acceptor sequences.
  • Figure 3 Sequence of HPV 18. Sequences of HPV 18 primers are indicated by underlines. Boxes indicate splice donors and acceptor sequences.
  • Figure 4 HPV 16 primer families. A variety of primers were tested by the ability to amplify total RNA from SiHa cells (infected with HPV 16). The reactions contained 10% DMSO and 15% sorbitol. The primers are indicated on the autoradiogram.
  • Figure 5 The effect of increasing the RNAse H concentration using HPV 16 primer families.
  • Figure 6 HPV 16 primer sensitivity.
  • Total RNA is titrated from 1, 0.1, 0.01, 0.001 attomoles of specific E6-7 RNA isolated from SiHa RNA. p. 32. N5.
  • Figure 7 Primer sensitivity using cells which contain HPV 18 DNA. From right to left is 104 to 10 cells. p34 N4.
  • Figure 8 An autoradiogram slotting 3SR reaction products.
  • RNAse titration was performed using primers 32-54 which amplified HPV 18 RNA.
  • the additives were 10% DMSO, 10% polyethylene glycol and 10% glycerol.
  • the cross reactivity using primers 29-15 using SiHa cell using these additives were included to determine if there was any cross reactivities of the reactions.
  • Figure 10 Autoradiogram of a 3SR reaction comparing primers 32-54 and 69-54.
  • the 3SR reaction using primers 69-54 contained either no additives (column 1) or 15% sorbitol (column 2).
  • the reactions using Primers 32-54 contained 10% polyethylene glycol (column 3). From top to bottom was a titration of RNAse H, 1-3 units per reaction.
  • FIG. 11 Co-amplification. Lane A used primers 136-73
  • Lane B used primers 136-91 (HPV 16) amplifying 5 amol of SiHa RNA using decreasing amounts of DMSO /sorbitol mixture. Lane C from top to bottom: 136-73 (HPV 16) and 54-69 (HPV 18), 136-91 and 54-69, and
  • Figure 12 HPV 16 plate optimization. Capture 245 temperature optimum. Absorbance values using CAP245 at different temperature ranges: 30°C, 40°C, 50°C, 60°C and 70°C. Each line represents a different detectors; DET 251, DET 252, and DET 254.
  • Figure 13 HPV 16 plate optimization. Capture 250 temperature optimum. Absorbance values using CAP250 at different temperature ranges: 30°C, 40°C, 50°C, 60°C and 70°C. Each line represents a different detectors; DET 251, DET 252, and DET 254.
  • Figure 14 Detector hybridization optimum using CAP 245. Detectors were hybridized using different temperature ranges: 30°C, 40°C, 50°C, 60°C and 70°C. Each line represents different detectors: DET 98, DET 251, DET 252, and DET 254.
  • Figure 15 Detector hybridization optimum using CAP 250. Detectors were hybridized using different temperature ranges: 30°C, 40°C, 50°C, 60°C and 70°C. Each line represents different detectors: DET 98, DET 251, DET 252, and DET 254.
  • Figure 16 HPV 16 plate assay. A comparison of captures 245,
  • Figure 17 HPV 16 detector performance. A comparison of all the detector oligos for HPV 16 using CAP 250. The detector names are listed in the bottom of each figure.
  • Figure 18 A comparison of detector lengths using CAP 250 in the enzyme probe assay.
  • DET 256 is a 17mer oligo and DET 257 is a 15mer oligo. The sequence was identical except that 2 bases were omitted for DET 257.
  • Figure 19 A comparison in absorbance values using different additives in the capture buffer. From left to right are duplicate wells using DET 255, DET 98 and DET 256. Columns 1-6 are 3SR products using primers 96-91. Columns 7-12 are 3SR products using primer 137-91 using different detectors. The additives are indicated on the left of the absorbance values. Rows 1 and 2 are plus and minus templates using 5% polyethylene glycol. Rows 3 and 4 are plus and minus templates using 1% BSA. Rows 5 and 6 are plus and minus templates using 5% PEG, 1% BSA. Rows 7 and 8 are the standard hybridization buffer using 0.1% polyvinylpyrrolidone, 5X SSC.
  • Figure 20 A comparison in absorbance values using different additives in the detection buffer. From left to right using different detectors: DET 256, DET 98, and DET 255. Columns 1, 5, and 9 contained the standard hybridization buffer 30% glycerol, 0.1% PVP, 1% BSA and 5X SSC. Columns 2, 6, and 10 contained 5% PEG, 0.1% PVP, and 5X SSC as the hybridization buffer. Columns 3, 7, and 11 contained 1% BSA, 0.1% PVP and 5X SSC as the hybridization buffer. Columns 4, 8, and 12 contained 5% PEG, 1% BSA, 0.1% PVP, and 5X SSC as the hybridization buffer.
  • Rows A and B are plus and minus templates using primers 96-91 which amplify SiHa RNA.
  • Rows C and D is plus and minus template using primers 136- 91 which amplify SiHa RNA.
  • Figure 21 Different primers sets which amplify HeLa RNA
  • Figure 22 Comparison of capture oligos for HPV 18 using the enzyme probe assay.
  • the 3SR product was amplified from HeLa RNA using primer 54-69.
  • Column 1 is substrate only.
  • Columns 2 and 3 are plus and minus templates using capture 56.
  • Columns 4 and 5 is plus and minus templates using capture 267. Rows indicate different detectors. Row A DET 59, Row B DET 260, Row C DET 262, Row D DET 268, Row E DET 269, and Row F DET 270.
  • Figure 23 Comparison of capture oligos for HPV 16 and HPV 18 using the enzyme probe assay.
  • the 3SR product was a co-amplification from HeLa and SiHa RNA using primers 136-91 (HPV 16) and 54-69 (HPV 18).
  • Figure 24 HPV 16 and HPV 18 EPA. The absorbance levels of a typical specimen. HPV 16 and HPV 18 were co-amplified using primers
  • Figure 25 Schematic of the Enzyme Probe Assay.
  • the capture oligo hybridizes to the amplified 3SR product either HPV 16 or HPV 18.
  • the complex is detected using HRP labeled oligonucleotide.
  • Figures 26 and 27 Autoradiographs of amplification products comparing yields of reaction performed at 50°C and at 42°C.
  • FIG 1 is a schematic drawing showing a generalized HPV 16 genome.
  • the heavy concentric lines indicate open reading frames.
  • Figures 2 and 3 locate the splice donor and acceptors for HPV 16 and 18 genes (indicated by boxes around the terminal two bases involved in the splice in the E6/E7 region).
  • the portion of the HPV 16 and 18 viral genomes coding for E6/E7 polypeptides are identified in the Sequence Listing as SEQ. ID. Nos. 1 and 2 respectively. This is a significant region of the genome since the proteins encoded are thought to be involved in degradation of the p53 suppressor protein, which regulates cell growth. Loss of p53 function is associated with malignancy.
  • expression of E6/E7 is diagnostic for cervical cancer or pre-malignant states.
  • 3SR self-sustained sequence replication
  • Polymerase chain reaction amplifies DNA, and while it may detect the presence of virus with great sensitivity, it is unsuitable for detecting gene expression.
  • the method of 3SR is fully described in Gingeras, et al., Ann. Biol. Clin., 48: 498 (1990), Guatelli, et al., PNAS, 87: 1874 (1990), and WO 90/06995. The methods described therein are followed herein except as noted, and define the procedure to be followed in the practice of the present invention.
  • the general 3SR amplification procedure as set forth in Gingeras et al. and Guatelli et al. involves the following steps: One hundred-microliter 3SR amplification reactions contained the target RNA, 40 mM Tris-HCl at pH 8.1, 20 mM MgCl 2 , 25 mM NaCl, 2 mM spermidine hydrochloride, 5 mM dithiothreitol, 80 ⁇ g/ml bovine serum albumin, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP, 4 mM ATP, 4 mM CTP, 4 mM GTP, 4 mM UTP, and 250 ng of each selected oligonucleotide primer.
  • 3SR is carried out as follows on HPV specimens: samples are obtained by vaginal lavage or cervical scrape. Messenger RNA is released by treatment with chaotrophic /phenol reagents and precipitated conventionally with ethanol. A preferred one step extraction utilizes RNAzol B (Cinna/Tiotecx Laboratories, Inc.) according to the manufacturer's instructions. The RNA is then dissolved in 3SR buffer, together with nucleotide and nucleoside triphosphates, primers, enzymes, and cofactors to carry out 3SR amplification. Reagents were obtained as follows:
  • oligonucleotides may be synthesized on a commercially available synthesizer such as a Milligen 8700 DNA synthesizer. Oligonucleotides which contained a 5' biotin may be synthesized using a biotin phosphoramidite (Glenn Research). Oligonucleotides which contain a 3' biotin may be synthesized using control pore glass containing a protected biotin (Glenn Research). Oligonucleotides which contain a 3' amine are conveniently synthesized using a amino-on control pore glass column (Glenn Research). Below is a list of oligonucleotides used in the development of HPV 16/18 enzyme probe assay of the present invention. All of the sequences are from left to right 5' to 3'. The oligonucleotide primers are also listed in the Sequence Listing as SEQ. ID. Nos. 3-31.
  • HPV53 GAA TGT GTG TAC TGCC AAG CAA CAG
  • HPV91 ACA GAG CTG CAA ACA ACT ATA CA 18
  • HPV92 AAT TTA ATA CGA CTC ACT ATA GGG
  • HPV101 AGA GCT GCA AAC AAC TAT ACA TG
  • HPV106 AAT TTA ATA CGA CTC ACT ATA GGG
  • HPV120 AAT TTA ATA CGA CTC ACT ATA GGG
  • HPV131 AAA CAA CTA TAC ATG ATA TAA TA 30
  • HPV136 AATTTAATACGA CTC ACT ATA GGG
  • Primer selection for high level amplification is basically a directed trial and error process.
  • a span of 400 bases was selected by designating the first 10-30 nucleotides at the 5' end of the E6 gene beginning with the ATG codon and counting off 400 bases, then selecting as primers the next 10-30 bases.
  • at least one of the primers must contain a promoter for transcription.
  • the bacteriophage T7 RNA polymerase binding site (SEQ. ID. No. 44), AAT TTA ATA CGA CTC ACT ATA GGG A, is preferred because of its strength and specificity.
  • the primer pairs are tested for their amplification efficiency.
  • the second primer position is held stationary and the first primer is moved arbitrarily 20 bases towards the second (thereby decreasing the interprimer span, e.g. the bases between the position of the 3' end of the first primer and the 5' end of the second primer, by 20 bases to 380 bases).
  • Fine tuning is accomplished by walking the primers from the best pairings by 2-5 base jumps.
  • Figure 4 gives primer families that amplify the HPV 16 E6-7. All primers amplified total RNA isolated from the SiHa cell line which contain the HPV 16 transcripts.
  • the reaction conditions include 7mM rNTPs, ImM dNTPs, 40mM Tris pH 8.1, 30mM MgCl 2 20mM KC1, 50mM dithiothreitol, 20 mM spermidine, 10% DMSO, 15% sorbitol, and 15pmol each priming oligonucleotide.
  • After pre-warming each tube at 42°C for 5 minutes 30 units of AMV-RT, 2 units RNAse H, and 250 units of T7 RNA polymerase were added as a cocktail to each reaction. The reaction was allowed to proceed for one hour at 42°C. A sample of the 3SR reaction was slotted onto nitrocellulose.
  • the nitrocellulose was baked for 45 minutes and then hybridized for 45 minutes using a type specific detection oligo.
  • An autoradiogram was generated by exposing the nitrocellulose to film for 45 minutes at -70°C.
  • the primer family for 120 is 29 and 90.
  • the primer family for 15 is 19, 20, 77, 53, and 89.
  • the primer family for primer 129 is 29, 74, 73, 118, 130, and 131.
  • the primer family for primer 136 is 91, 29, 90, 74, 73, 130, 131, and 118.
  • the primer family for primer 137 is 29, 90, 74, 73, 131, and 118.
  • Figure 5 illustrates the effect of titrating the RNAse H HPV 16 primer families.
  • the 3SR reaction conditions are identical as described in figure 4 except the DMSO and sorbitol were omitted from the reaction. Ten microliters were slotted onto nitrocellulose then baked and probed with a type specific detection oligo (HPV55).
  • the primer family for primer 93 is 73 and 91.
  • the optimal RNAse H needed for the reaction using these two primer pairs is between 1 and 2 units.
  • the primer family 95 is 101 and 91. These primer sets do not appear to be sensitive to different RNAse H concentrations.
  • a single primer set was defined for primer 92; 92-91, primer 94; 94-91, and primer 85; 85-77.
  • the primer family for primer 96 is 73 and 91.
  • primers 96-73, 96-91, and 94-91 were tested using a titration of E6-7 isolated from SiHa cells. Once each primer set has been defined and optimized the sensitivity can be measured by amplifying decreasing amounts of RNA from control cells (figure 6).
  • the 3SR reaction conditions are identical to those described in figure 4 except, using primers 96-73 the DMSO was included and the sorbitol was omitted, and using primers 94-91 only 10% sorbitol was included.
  • Figures 7-10 describe the primers used to amplify HPV 18 E6- 7.
  • the primer family for primer 54 is 32, 69, and 70.
  • Primers 48 and 32 also amplify HeLa RNA.
  • Primers 54-32 and 54-48 both require the addition of additives 10% polyethylene glycol or DMSO and sorbitol to the 3SR reaction.
  • Primers 54-69 do not require the addition of additives for successful amplification.
  • Additional primer families for primer 214 is 69, 244, 214, and 70 all which require additives to the amplification reaction.
  • Co-amplification Once primers have been selected for both HPV 16 and HPV 18 a co-amplification of both targets is required for clinical use. Co-amplification is required because only a single specimen is obtained. This can be done not only for HPV 16 or HPV 18, but also can be applied to a plurality of HPV types including but not limited to HPV 31, 33, and 35, as well as any other types that prove to be oncogenic. It is not practical to split a single specimen for two independent reactions.
  • Figure 11 is a duplicate blot which is probed with a 16 and 18 type specific detection probe. Lane C demonstrates the cross reactivity of amplifying two independent targets.
  • Capture and Detection Probes Because it is impractical to incubate the plate in elevated temperatures the detector should produce maximum signal at room temperature. Many times uneven temperatures across a microwell can cause differences in hybridization thereby causing variability of absorbance values.
  • the format of the plate affects the performance of the assay. Incubating both capture and detector probes simultaneously rather than capturing the 3SR product first and detecting in a separate incubation step affects the relative OD values. There are disadvantages of co-incubation of both capture and detection probes. In high template concentration, the 3SR reaction produces very high product concentrations. When the capture is incubated to the target in one step then applied to the microwell and allowed to bind, excess target is subsequently washed away. The detection probe is then applied which only hybridizes to the capture 3SR target.
  • FIG. 12 and 13 define the optimum temperature of hybridization for HPV 16 capture oligonucleotide.
  • the 3SR product is diluted 1:10,000 to reduce the absorbance levels thereby allowing differences of different detection probes to become more pronounced.
  • the hybridization reaction contain 50 ⁇ l of the diluted 3SR product in 0.1% PVP, 2X SSC, and 4 pmol capture oligonucleotide. The reaction was incubated at different temperatures ranging from room temperature to 70°C.
  • the reaction proceeded in the microwell for 20 minutes and the well washed 3 times with 2X SSC (0.6 M NaCl, 0.06 M Na citrate pH 7.0), 0.05% Tween 20®, and 0.01% ThimersolTM.
  • the detection probe was added and incubated for 30 minutes at room temperature.
  • the microwell was again washed 3 times with 2X SSC, 0.5% Tween 20, and 0.01% Thimersol.
  • Substrate for the horseradish peroxidase enzyme 3', 3', 5', 5', tetra methyl benzidine and hydrogen peroxide was added to each well and allowed to develop for 15 minutes at room temperature. The reaction was stopped by the addition of 1 M phosphoric acid and read at 450 nm.
  • the optimum temperature of hybridization for capture 245 is between 50°C and 60°C.
  • the signal remains relatively constant at 70°C but thermal degradation of the RNA is a concern at this temperature.
  • Capture 250 hybridization optimum is between 50°C and 60°C.
  • a variety of detection probes should be tested because the optimum temperatures for hybridization of the detection probes must be empirically determined. Once the capture oligo temperature optimum has been defined, the same experiments must be repeated using different probes.
  • Figures 14 and 15 define the detector optimum.
  • CAP 250 and CAP 245 produced the highest absorbance values when hybridizing DET 251 at room temperature.
  • the reaction was performed as described in figure 13.
  • the following is a list of useful detection, capture probes, and positive hybridization control probes.
  • the detection, capture and positive hybridization control probes are also listed in the Sequence Listing as SEQ. ID. Nos. 32-43.
  • BIOTIN 34 CAP 253 TGT ATT AAC TGT CAA AAG CCA AAA AAA
  • BIOTIN 36 CAP267 GTG CCT GCG GTG CCA GAA AAA AAA
  • Figure 16 is a comparison of all the best performing capture probes using 4 different detection probes.
  • the capture probes were hybridized to the 3SR product at the temperature optima for 30 minutes in
  • CAP 250 produces the highest signal when amplifying SiHa cells; however, CAP 250 only can capture two of the three spliced E6 RNA's. Several other capture probes were investigated and CAP 265 captures all three E6 transcripts. Each cell line splices E6 at different rates. CAP 265 was chosen because clinical specimens may be heterogenous in splicing E6.
  • FIG 17 is a comparison of all the detection probes for HPV 16.
  • DET 256 produces the highest absorbance values in the present assay.
  • Two detection probes were synthesized for illustration. The first a 17mer and the second a 15mer to define the minimum number of bases needed for efficient hybridization. The minimum length a detector oligo can be is about 17 bases (figure 18). Please note that best results are achieved when the signal enzyme is conjugated to the oligonucleotide at the 3' end.
  • Various additives in the capture buffer were performed with little increase in the relative absorbance in the plate assay (figure 19). When these same additives were added to the detection buffer the signal was more than doubled (figure 20).
  • the capture oligonucleotide is biotinylated through 3' or 5' terminal labeling by conventional techniques. It has been empirically determined for the probes studied to date that biotinylating the capture probe at the 3' terminus is more efficient in immobilizing the probe hybridized to sample target sequence.
  • the solid phase is coated with streptavidin, so that when the hybridized capture-sample sequence complex is brought into contact with it, the reaction between streptavidin and biotin takes place.
  • the solid phase is preferably the inner surface of microtiter tray wells, but any solid phase separation system known to the art is satisfactory including but not limited to polystyrene beads, magnetic microparticles, test strips of plastic or metal, dipsticks, columns packed with a variety of materials, etc.
  • the fluid phase capture method of the present invention is expected to give enhanced results with solid supports made of plastic because of the especially low capture efficiencies with plastic supports in conventional assays.
  • Any signal-generating enzyme or other reporter or tracer system capable of being conjugated covalently or electrostatically to a oligonucleotide without hindering its hybridizing to a complementary sequence is contemplated in the present assay.
  • Horseradish peroxidase is preferred, but alkaline phosphatase and synthetic fluorogenic and chromogenic molecule hydrolyzing enzymes may also be employed.
  • Non- isotopic reporter /tracer systems are preferred over radioactive tracers because of environmental and stability considerations.
  • Figure 26 compares amplification reactions performed using the standard 3SR reaction conditions (42°C) with amplification reactions performed at an elevated temperature (50°C).
  • the assays used the primer sets 136-91 (HPV 16) and 54-69 (HPV 18) together and separately.
  • the standard 3SR reaction conditions were 40 mM Tris-HCl, pH 8.1; 30 mM MgCl 2 ; 20 mM KC1; 10 mM dithiothreitol; 4 mM spermidine; 15 pmole each priming oligonucleotide; 1 mM dNTP's; 7 mM rNTP's; 30 units AMV reverse transcriptase; 2 units RNAse H; and 1000 units T7 RNA polymerase.
  • the reaction was incubated for 1 hour at 42°C.
  • the elevated temperature reaction conditions were 40 mM Tris acetate, pH 8.1; 30 mM Mg acetate; 10 mM dithiothreitol; 100 mM potassium glutamate, pH 8.1; 1 mM dNTP's; 6 mM rNTP's; 15% sorbitol; 30 units AMV reverse transcriptase; 2 units RNAse H; and 1000 units T7 RNA polymerase.
  • the reaction was incubated for 1 hour at 50 °C.
  • 1/lOth of the amplification products were denatured in 90 ⁇ l of 7.4% formaldehyde and 10X SSC in a 65°C water bath for 10 minutes and quick-chilled on ice for at least 1 minute.
  • BA-85 nitrocellulose was pre-wetted with water and then with 10X SSC.
  • the denatured amplification samples were applied to a slot blot apparatus containing the pre-wetted nitrocellulose and the samples were drawn onto the nitrocellulose using a vacuum.
  • the filter was then baked for 45 minutes at 80°C and hybridized with a type-specific oligonucleotide specific for HPV 18 (DET59) or HPV 16 (DET98).
  • the hybridization solution contains 6X SSC; 10X Denhardts; 10 mM Tris, pH 7.4; 0.2 mg/ml sheared salmon sperm DNA; and 1% SDS.
  • Figures 26 and 27 depict a comparison of the amplification yields of reactions performed at 50°C and at 42°C.
  • the amplification reactions in column 1 used the HPV 16 primers 136-91
  • the reactions in column 2 used the HPV 18 primers 54-69
  • the reactions in column 3 used a combination of the HPV 16 and HPV 18 primers 136-91 and 54-69.
  • the target sequence was a mixture of 5 amol each of SiHa cell (infected with HPV 16) and HeLa cell (infected with HPV 18) RNA.
  • Rows 1-4 contained sorbitol concentrations of 15%, 10%, 5% and 0% respectively; row 5 was a minus template reaction using 15% sorbitol; row 6 was blank; and rows 7-11 contained sorbitol concentrations of 15%, 10%, 5% and 0% respectively. Rows 1-5 were incubated at 50°C and rows 7-11 were incubated at 42°C.
  • the amplification products in figure 26 were probed with DET 98 which is specific for HPV 16.
  • the amplification products in figure 27 were probed with DET 59 which is specific for HPV 18.
  • Figure 26 depicts that the bands were much stronger at the
  • ORGANISM Papaoviridae, Human papilloma virus
  • ORGANISM Papovaviridae, Human papilloma virus
  • NAME/KEY Portion of viral genome coding for E6/E7 polypeptides.
  • MOLECULE TYPE (A) DESCRIPTION: Other nucleic acid, synthetic
  • MOLECULE TYPE (A) DESCRIPTION: Other nucleic acid, synthetic
  • MOLECULE TYPE (A) DESCRIPTION: Other nucleic acid, synthetic
  • MOLECULE TYPE (A) DESCRIPTION: Other nucleic acid, synthetic
  • FEATURE (A) NAME/KEY: HPV120. Phage T7 RNA polymerase binding site at 5 'end, followed by HPV-16/18 sequence.
  • MOLECULE TYPE (A) DESCRIPTION: Other nucleic acid, synthetic
  • NAME/KEY HPV136. Phage T7 RNA polymerase binding site at 5 'end, followed by HPV-16/18 sequence.
  • MOLECULE TYPE (A) DESCRIPTION: Other nucleic acid, synthetic
  • MOLECULE TYPE (A) DESCRIPTION: Other nucleic acid, synthetic
  • MOLECULE TYPE (A) DESCRIPTION: Other nucleic acid, synthetic

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Abstract

Méthode de détection en deux étapes se basant sur une sonde d'hybridation d'acide nucléique de certains types de papillomavirus chez l'homme (HPV) associés à une dysplasie et à une malignité des cellules cervicales. Ladite méthode comprend une étape d'hybridation à capture en phase liquide, dans laquelle on hybride à un réactif de capture biotinylé un ARN messager amplifié de gènes spécifiques E6/E7 obtenu à partir d'un spécimen biologique, de façon à préparer un complexe; la fixation du complexe de réactif de capture à une phase solide par réaction avec de la streptavidine immobilisée; une deuxième étape d'hybridation dans laquelle une sonde de détection conjugée à un enzyme spécifique au virus s'hybride avec le complexe d'ARN messager amplifié; enfin, la détection du complexe sonde de détection par production couleur ou fluorogène suivant le lavage de la phase solide et l'apport d'un substrat approprié chromogène ou fluorogène. La méthode de détection présente une sensibilité amplifiée par rapport aux procédés classiques, ainsi qu'une spécificité d'expression réelle d'oncogènes de HPV dans des spécimens cervicaux, n'indiquant pas uniquement la présence d'un virus.
PCT/US1994/005085 1993-05-06 1994-05-06 Methode de detection du papillomavirus chez l'homme WO1994026934A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP94917931A EP0655091A1 (fr) 1993-05-06 1994-05-06 Methode de detection du papillomavirus chez l'homme
JP6525595A JPH07508891A (ja) 1993-05-06 1994-05-06 ヒト乳頭種ウイルス検出アッセイ
AU69456/94A AU6945694A (en) 1993-05-06 1994-05-06 Human papillomavirus detection assay
CA002139623A CA2139623A1 (fr) 1993-05-06 1994-05-06 Test de detection des papillomavirus chez l'humain

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US5892093A 1993-05-06 1993-05-06
US08/058,920 1993-05-06

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WO1994026934A2 true WO1994026934A2 (fr) 1994-11-24
WO1994026934A3 WO1994026934A3 (fr) 1995-01-26

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FR2737502A1 (fr) * 1995-07-31 1997-02-07 Genset Sa Procede de detection d'acides nucleiques utilisant des sondes nucleotidiques permettant a la fois une capture specifique et une detection
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EP0821059A2 (fr) * 1996-07-25 1998-01-28 The Institute Of Physical & Chemical Research Méthode pour transcription inverse
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US6447995B1 (en) 2000-10-04 2002-09-10 Genvec, Inc. Utilizing intrinsic fluorescence to detect adenovirus
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WO2003057914A2 (fr) * 2002-01-07 2003-07-17 Norchip A/S Methode permettant de detecter l'arn messager du papillomavirus humain
AU766744B2 (en) * 1995-11-15 2003-10-23 Gen-Probe Incorporated Nucleic acid probes complementary to human papillomavirus nucleic acid and related methods and kits
EP1397489A2 (fr) * 2000-04-13 2004-03-17 Medical University of South Carolina Agents toxiques, riboyzmes, andyzmes et oligonucleotides antisens specifiques des tissus et des pathogenes et procede d'utilisation associe
EP1422298A3 (fr) * 1999-07-09 2004-07-21 Gen-Probe Incorporated Détection de l'HIV-1 par amplification des acides nucléiques
WO2006084155A1 (fr) * 2005-02-02 2006-08-10 Patterson Bruce K Dosage d'arnm d'e6, e7 d'hpv et methodes d'utilisation associees
US20070111960A1 (en) * 2005-03-04 2007-05-17 Advandx, Inc. High affinity probes for analysis of human papillomavirus expression
US7354719B2 (en) 2004-12-08 2008-04-08 Gen-Probe Incorporated Detection of nucleic acids from multiple types of human papillomaviruses
US7445930B2 (en) 1996-11-20 2008-11-04 Introgen Therapeutics Inc. Method for the production and purification of adenoviral vectors
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US20120129238A1 (en) * 2010-10-22 2012-05-24 Bio-Rad Laboratories, Inc. Reverse transcriptase mixtures with improved storage stability
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WO1996026293A3 (fr) * 1995-02-24 1996-12-12 Deutsches Krebsforsch Procede de detection precoce de carcinomes associes au vph ou de dysplasies graves provoquees par le vph
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DE19526717A1 (de) * 1995-07-21 1997-01-23 Florian Dr Med Heirler Verfahren zur Diagnose des Zervixkarzinoms
WO1997005277A1 (fr) * 1995-07-31 1997-02-13 Genset Procede de detection d'acides nucleiques utilisant des sondes nucleotidiques permettant a la fois une capture specifique et une detection
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US6228580B1 (en) 1995-07-31 2001-05-08 Genset Nucleic acid detection method using nucleotide probes enabling both specific capture and detection
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CA2139623A1 (fr) 1994-11-24
AU6945694A (en) 1994-12-12
EP0655091A1 (fr) 1995-05-31
WO1994026934A3 (fr) 1995-01-26
JPH07508891A (ja) 1995-10-05

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