Classifications

• • 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
  • C12Q1/706—Specific hybridization probes for hepatitis
  • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
• • 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

Definitions

• the invention relates to the area of detecting polynucleotide analytes. More particularly, the invention relates to diagnostic assays based on the detection of specific nucleotide sequences.
• PCR polymerase chain reaction
• PCR has inherent problems and limitations.
• Very small amounts of the amplified target sequence of which up to 10 9 copies can be present in a single PCR solution, can contaminate laboratory equipment or reagents.
• the PCR product can even spread as airborne droplets in areas of sample or reagent preparation. This contaminating DNA can then serve as a template for further amplification, resulting in false positive results in subsequent samples.
• a common precaution against this type of error is the establishment of procedures to physically separate the steps of PCR reactions in a laboratory and/or the routine use of chemical and enzymatic methods for inactivating PCR products.
• PCR-based diagnostic methods Another problem with PCR-based diagnostic methods is that the clinical relevance of a positive PCR result is questionable when small numbers of pathogenic organisms are present in samples from persons who are clinically unaffected. Further, because PCR amplifies only a portion of the genome of an infectious agent, another source of error is the detection of nonviable organisms. In such instances, detection of cDNA by reverse-transcription PCR of messenger RNA encoded by the pathogenic organism could be misinterpreted as evidence of active infection. Finally, because PCR uses two primers to achieve exponential amplification, the method is sensitive to genetic variability of the target sequence. Thus, there is a need in the art for reliable and convenient methods which can sensitively and specifically detect single-stranded DNA analytes in a biological sample.
• One embodiment of the invention is a method of detecting the presence of a single-stranded polynucleotide analyte in a biological sample.
• a polynucleotide molecule on a solid support is detected.
• the polynucleotide molecule comprises a single-stranded polynucleotide analyte and one or more single-stranded polynucleotide probes which specifically hybridize to the single-stranded polynucleotide analyte to form one or more first portions of the polynucleotide molecule which are double-stranded.
• At least one of the single-stranded polynucleotide probes is bound to the solid support. Detection of the first portion of the polynucleotide molecule on the solid support indicates the presence of the single- stranded polynucleotide analyte in the biological sample.
• kits for detecting a single-stranded DNA analyte in a biological sample comprising at least one DNA probe which is single-stranded, a solid support, and a monoclonal antibody which specifically binds to double-stranded DNA.
• the DNA probe comprises a first binding moiety.
• the solid support comprises a second binding moiety. The first and second binding moieties specifically bind to each other.
• kits for detecting a single- stranded DNA analyte in a biological sample comprises a solid support and a monoclonal antibody which specifically binds to double-stranded DNA.
• the solid support comprises one or more single-stranded DNA probes.
• Even another embodiment of the invention is a single-stranded DNA primer which consists of a sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOS: 1, 2, 3, 5, 8, 10, and 12.
• the present invention thus provides the art with a method of detecting a single-stranded polynucleotide analyte in a biological sample.
• the method is sensitive, specific, and reliable, and can be used in manual or automated diagnostic or screening assays.
• Figure 1 demonstrates detection of CMV particles in serum using Amplification Independent DNA Assay (AID A).
• Figure 2 demonstrates the increase in sensitivity obtained after carrying out a polymerase elongation step after hybrid capture.
• Figure 3 shows the results of an AID A test on the sera of control and HIV- positive patients.
• the present invention solves problems inherent with exponential amplification methods by using a single polynucleotide probe to both capture a single-stranded polynucleotide analyte and to generate a detection signal.
• the method provides a sensitive and specific method of detecting a particular single-stranded polynucleotide analyte in a biological sample without need to resort to the elaborate and expensive procedures required with PCR-based methods.
• the method is capable of detecting a single-stranded DNA analyte present at a concentration as low as 0.1 fg/ ⁇ l.
• the single-stranded polynucleotide analyte can be DNA or RNA, preferably genomic or cDNA. If the presence of a DNA virus in a biological sample is suspected, specific sequences in the viral genome can be detected after extraction of genomic DNA from serum or whole blood (see Example 8, below).
• a single- stranded DNA analyte can be generated in the biological sample from a double- stranded starting material, for example, by heating or chemically treating the biological sample to denature double-stranded DNA. If the presence of an RNA virus is suspected, the RNA viral genome can be converted into single-stranded DNA using reverse transcriptase, as is known in the art (see Example 2, below). Similarly, the presence of any mRNA can be detected by first converting the mRNA into single- stranded DNA.
• Methods of the invention can be used to detect the presence of pathogens, such as a bacteria, viruses such as hepatitis C (HCN), hepatitis B (HBN), hepatitis G (HGV), or human immunodeficiency virus (HIN), fungi, protozoa, parasites, or mycoplasma. Any organism which contains R ⁇ A or D ⁇ A can be detected.
• the method can be used to detect the presence of genetic mutations which have diagnostic or prognostic value. Pathogen contamination of food and drink supplies can also be detected using the method.
• the biological samples used for diagnostic purposes can be, for example, samples such as tissue or cellular extracts, whole blood, serum, or plasma.
• Blood samples can be obtained by venepuncture or by accessing capillary veins of the finger or heel of a postnatal human. Tissue or cell samples can be obtained using appropriate biopsy methods. Prenatal samples of fetal blood or tissue can also be tested. Biological samples used for analytical purposes can be those described above or can be, for example, samples of food, drink, or bodies of water, such as ponds, rivers, lakes, or pools.
• the presence of a single-stranded polynucleotide analyte in a biological sample is indicated by detecting a double-stranded polynucleotide molecule on a solid support.
• the double-stranded polynucleotide molecule is formed by specific hybridization of the single-stranded polynucleotide analyte with one or more single- stranded polynucleotide probes. After hybridization, all or a portion of the single- stranded polynucleotide analyte can be double-stranded. At least one of the probes is bound to the solid support and thus is used both to capture the single-stranded polynucleotide analyte and to form a double-stranded polynucleotide portion which is then detected.
• AID A Amplification Independent DNA Assay
• the presence of a single-stranded polynucleotide analyte, preferably a single- stranded DNA analyte, present in very low concentration in a biological sample can be detected independent of analyte amplification.
• the only amplification which may occur in AID A is amplification of the number of regions of the single- stranded analyte which can be detected using a reagent which specifically binds to double-stranded polynucleotide molecules, such as an anti-double-stranded DNA antibody.
• This procedure is highly innovative because it eliminates problems related to analyte amplification, such as cost and contamination, and also provides high levels of sensitivity.
• the AIDA assay can be carried out in any practical sample volume. Most conveniently, the AIDA assay is carried out in a 0.05 to 5 ml sample, preferably in a 0.1 to 1 ml sample.
• a biological sample is contacted with one or more polynucleotide probes, preferably DNA probes, which are single-stranded and complementary to one or more regions of the single-stranded DNA analyte.
• a DNA probe is complementary to a single-stranded DNA analyte if its nucleotide sequence will form hydrogen-bonded base pairs with a nucleotide sequence in the single- stranded DNA analyte.
• DNA probes which are complementary to any known nucleotide sequence can be synthesized chemically, using an automated oligonucleotide synthesizer, such as the PCR Mate - EP 391 DNA synthesizer
• DNA probe is at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length, and DNA probes of at least 50 nucleotides are preferred. However, any probe, regardless of length or sequence, can be used.
• Preferred DNA probes for use in the present invention are those which comprise the sequences shown in SEQ ID NOS:6, 9, 13, and 18.
• the nucleotide sequence shown in SEQ ID NO: 6 is complementary to a portion of a hepatitis C genome.
• the nucleotide sequence shown in SEQ ID NO: 9 is complementary to a portion of a hepatitis B genome.
• the nucleotide sequence shown in SEQ ID NO: 13 is complementary to a portion of a hepatitis G genome.
• the nucleotide sequence shown in SEQ ID NO: 18 is complementary to a portion of an HIV genome.
• the biological sample is contacted with the single-stranded polynucleotide probe in an aqueous solution.
• the solution can contain certain agents, such as polyvinylpyrrolidone, bovine serum albumin, the synthetic sucrose polymer "FICOLL 400,” salmon sperm DNA, and yeast RNA, to decrease nonspecific binding of the probe to proteins, polysaccharides, and nucleic acids.
• the step of contacting is carried out under conditions where single-stranded polynucleotide probes hybridize to complementary single-stranded polynucleotide analytes to form double-stranded polynucleotide molecules.
• Variables involved in determining hybridization conditions include probe length and concentration, concentration of single-stranded polynucleotide analyte, temperature, salt concentration, and the relative percentage of G-C versus A-T bonds which can be formed between the polynucleotide probe and the single-stranded polynucleotide analyte.
• Conditions suitable for hybridizing a particular probe-analyte pair can readily be determined by one of skill in the art. Basic manuals of recombinant DNA techniques, such as Sambrook et al, MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (Cold Spring Harbor Press, Cold Spring Harbor, New York), can be referred to for this purpose.
• Single-stranded polynucleotide molecules which do not specifically hybridize to the single-stranded polynucleotide probes employed can be eliminated, for example by one or more washing steps.
• At least one of the polynucleotide probes which hybridize to the single- stranded polynucleotide analyte is used to affix the double-stranded polynucleotide molecules to a solid support.
• the single-stranded polynucleotide probe can be attached to the solid support prior to or after hybridization with the single-stranded polynucleotide analyte, as is desired.
• the probe can comprise a first binding moiety, such as biotin.
• a biotin moiety can be bound to the 5' end of a DNA probe, for example, during automated synthesis by using Biotin-Phosphoramidite (Amersham), resulting in a biotinylated DNA probe.
• the solid support then comprises a second binding moiety, such as avidin or streptavidin.
• the first binding moiety can be avidin or streptavidin and the second moiety can be biotin.
• Other specific binding pairs such as antibody-antigen pairs, can be used to bind the probe to the solid support. Any two binding partners can be used which specifically bind to each other with a K D less than 10 '7 , lO '8 , 10 "9 , or preferably 10 "12 .
• the probe can be covalently bound to the solid support, for example by use of a covalent cross-linker such as l-(p-azidosalicylamido)-4-
• the solid support can be any surface to which the single-stranded probe can be attached.
• Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, or particles such as beads, including but not limited to latex, polystyrene, or glass beads.
• the efficiency of the method is increased if particles are used as the solid support.
• the particles can be placed in a tube for use as a fixed or removable cartridge or in a microtiter well, for use in either manual or automated assays. Detection of the presence of a double-stranded polynucleotide molecule on the solid support indicates the presence of the single-stranded polynucleotide analyte in the biological sample.
• a reagent which specifically binds to double-stranded polynucleotide molecules, particularly double-stranded DNA molecules can be employed for this purpose.
• reagents include, but are not limited to, proteins (such as antibodies, antibody fragments, and DNA binding proteins) and dyes specific for double-stranded DNA, such as ethidium bromide.
• the reagent is an antibody, such as a polyclonal or monoclonal antibody, or an antibody fragment, such as an Fab, F(ab)' 2 , or a single-chain antibody.
• the antibody has an affinity for double-stranded DNA molecules at least 100-, 500-, 1,000- or 10,000-fold greater than for single-stranded DNA molecules.
• a monoclonal antibody with these properties can be produced, for example, by conventional procedures for generating antibody-secreting hybridomas, using spleen cells from an animal which is susceptible to autoimmune disease, such as an MRL/lpr mouse.
• the bound antibody can be directly or indirectly coupled to a reporter system, in which a detectable signal is generated which is proportional to the amount of double-stranded DNA which is bound to the solid support.
• the antibody can comprise a detectable label, such as a radioactive, fluorescent, chemiluminescent, or colloidal gold label. Methods for detecting these labels, such as spectrophotometry, autoradiography, nephelometry, fluorimetry, or flow cytometry, are well known in the art.
• the antibody can be detected using an indirect immunochemical method, for example, by adding peroxidase-or alkaline phosphatase-labeled protein A after binding the double-stranded DNA antibody and carrying out a chromogenic reaction, or by use of a second antibody which is labeled and immunoreactive with the first antibody.
• the presence of antibody bound to double-stranded DNA molecules can be detected by monitoring changes in electrical conductance of a solution in contact with stranded DNA molecules, as is known in the art.
• the antibody and detection system used are capable of detecting double-stranded DNA molecules which are present in the biological sample at a concentration of less than 0.3 pg/ ⁇ l.
• double-stranded DNA molecules which are present in the biological sample at a concentration of less than 5 fg ⁇ l . less than 1 fg/ ⁇ l, less than 0.5 fg/ ⁇ l, or less than 0.1 fg/ ⁇ l can be detected.
• a preferred detection system is the ETI-2 system, available from DiaSorin and described in Example 1, below.
• the method of detecting the amount of antibody bound to double- stranded DNA molecules provides a signal which is at least 2-, 5-, 10-, 20-, or 50- fold higher than a background signal.
• the sensitivity of the detection step can be increased, for example, by providing additional binding epitopes for the anti-double- stranded DNA antibody. Additional binding epitopes can be provided in the hybridization step, by adding additional single-stranded DNA probes which are complementary to the single-stranded DNA analyte but whose nucleotide sequence does not overlap that of the DNA probe used to affix the analyte to the solid support.
• Additional binding epitopes can also be provided by carrying out a polymerase elongation step, either before or after the analyte is affixed to the solid support, before the addition of the antibody.
• a polymerase elongation step either before or after the analyte is affixed to the solid support, before the addition of the antibody.
• single stranded portions of the DNA analyte can be converted to double-stranded DNA, creating a considerable increase in the number of epitopes which can be recognized by the anti-double stranded DNA antibody.
• any conventional, non-thermostable polymerase such as T4, T7, or Klenow, can be used.
• it is also possible to provide additional binding epitopes by coupling additional double-stranded DNA, either in linear form or as dendrimers, to the single- stranded DNA probe.
• Detection of double-stranded polynucleotide molecules can be either qualitative or quantitative. Qualification can be accomplished, for example, by comparing the amount of detectable label on an antibody which specifically binds to a double-stranded DNA with a standard curve or by providing an internal standard against which to measure the amount of antibody which is bound to double-stranded
• the AIDA method provides a unique tool for simultaneously detecting the presence of two or more different single-stranded polynucleotide analytes in a single sample using a single reaction. This ability is useful, for example, to screen erythrocyte concentrates prepared from donated blood for the presence of HTV, hepatitis B, hepatitis G, and hepatitis C viral sequences, in order to avoid contamination which can occur during the window period.
• the biological sample is contacted with a mixture comprising single-stranded DNA probes specific for each single-stranded DNA analyte to be detected.
• a biological sample which provides a positive detection signal when screened can then be analyzed with individual probes to identify the contaminating single-stranded DNA analyte.
• additional copies of a single-stranded DNA analyte are synthesized to enrich the biological sample for the single-stranded DNA analyte.
• the size of the biological sample can thus be smaller than the biological sample used in the AIDA method above.
• This method is termed LEDIA (Linearly Enriched DNA Immunoassay).
• the LEDIA method can be performed using a biological sample of 100 ⁇ l.
• Additional copies of the single-stranded DNA analyte can be synthesized, for example, using a thermostable DNA polymerase isolated from an organism such as Pyrococcus furiosus or Thermus aquaticus. Any commercially available thermal cycler can be used.
• the step of synthesizing employs one single-stranded DNA primer so that additional copies of the complement of the single-stranded DNA analyte are not synthesized.
• the kinetics of enrichment of the biological sample for the single-stranded DNA analyte are linear.
• fewer than 50 polymerization cycles are employed.
• fewer than 49, 48, 47,46, 45, 40, 39, 38, or 30 polymerization cycles are employed.
• Primers for use in the LEDIA method must be specifically identified by routine testing in a LEDIA assay. Testing of several possible primers generally results in identification of a primer which is suitable for enriching a biological sample for a particular single-stranded DNA analyte.
• Examples of single-stranded primers which are suitable for use in the LEDIA method are primers which comprise one of the nucleotide sequences shown in SEQ ID NOS:l, 2, 3, 4, 5, 8, or 12. Primers with the nucleotide sequences shown in SEQ ID NOS: 1-5 can be used to enrich a biological sample for portions of a hepatitis C genome.
• Primers with the nucleotide sequence shown in SEQ ID NO: 8 can be used to enrich a biological sample for a portion of a hepatitis B genome.
• Primers with the nucleotide sequence shown in SEQ ID NO: 12 can be used to enrich a biological sample for a portion of a hepatitis G genome.
• Detection of a single-stranded DNA analyte using the LEDIA method does not require any chemical modification of the analyte. Furthermore, the LEDIA method avoids the risk of contamination which is inherent in exponential amplification techniques such as PCR. Because the enriched strand contains no sequences which are complementary to the DNA probe, the enriched strand cannot serve as a template for further reactions. The risk of contamination is therefore minimized. The LEDIA method therefore can sensitively and specifically detect a single-stranded DNA analyte without the occurrence of false positives which frequently occur with exponential amplification techniques. This feature of the LEDIA method avoids the need to set up cumbersome and often expensive procedures for physically isolating different steps of the reaction, such as treatment of samples, preparation of reagents, execution of the reaction, and analysis of the end-products of the reaction.
• a further advantage of the single-primer-based LEDIA method is that it is less sensitive to genetic variability of the single-stranded DNA analyte than methods based on the use of two primers, such as PCR. This feature of LEDIA is particularly relevant for detection of viruses which have high genetic variability, such as HIV.
• the LEDIA method is also particularly well-suited for quantitative applications. Because the enrichment of the biological sample for the single-stranded DNA analyte is linear rather than exponential, results can be reliably compared, for example, to a standard curve.
• the LEDIA method can be used to detect two or more different single-stranded DNA analytes in the same biological sample.
• Two or more different single-stranded DNA primers can be used to enrich the biological sample for two or more single-stranded DNA analytes.
• primers which can be used for linear enrichment of portions of hepatitis B, hepatitis C, hepatitis G, and HIV genomes can be used to amplify each of these analytes in one biological sample.
• kits which can be used for diagnosis or to screen a biological sample for the presence of one or more polynucleotide analytes.
• Kits which comprise one or more single-stranded DNA probes, a solid support, and a monoclonal antibody can be used to detect a single-stranded DNA analyte in a biological sample.
• the DNA probe can comprises a first binding moiety and the solid support can comprises a second binding moiety which specifically binds to the first binding moiety, as described above.
• the solid support can comprise one or more single-stranded DNA probes.
• Kits can additionally comprise written instructions for detecting a single- stranded DNA analyte using the AIDA method.
• a thermostable DNA polymerase such as a polymerase isolated from Thermus aquaticus or Pyrococcus furiosus, and a primer for linear enrichment of a single-stranded DNA analyte can be included in a kit, together with written instructions for detecting the single-stranded polynucleotide using the LEDIA method.
• Kits which can be used to detect two or more distinct single-stranded DNA analytes in a single biological sample can be provided by including additional DNA probes and single-stranded DNA primers.
• the AIDA and LEDIA methods can also be employed in partially or fully automated assays.
• An automated microwell plate reader can be used to carry out the detection step of the method.
• an automated plate processor can be used to carry out the entire method.
• solid supports such as beads cn be placed in a tube or cartridge, as described above.
• Example 1 demonstrates detection of CMV-DNA in sera by AIDA. Extraction of DNA.
• CMV particles TOWNE strain, AD 169 were obtained from ABI, Rivers Park, Guilford Road, Columbia, MD 21048. The viral particle count was 3.85 billion/ml.
• CMV particles were serially diluted in 200 ⁇ l of normal serum obtained from a CMV negative blood donor. DNA was extracted from the spiked serum using a QIAamp Blood kit by Qiagen (Cat. No. 29104) according to the manufacture's instructions. Qiagen protease (25 ⁇ l) was added to a 1.5 ml centrifuge tube with 150 ⁇ l of serum, and the volume was adjusted to 200 ⁇ l with
• the column was placed in a clean 2 ml collection tube, and the collection tube containing the filtrate was discarded.
• the QIAamp spin column was carefully opened and another 500 ⁇ l of Buffer AW was added.
• the column was again centrifuged at full speed for 3 minutes. After centrifiigation, the column was placed in a clean 1.5 ml microfuge tube, and the collection tube containing the filtrate was discarded. Hybridization.
• the DNA was eluted from the column with 50 ⁇ l of buffer AE preheated to 70 °C.
• the samples were incubated at 70 °C for 5 minutes in a heating block and then centrifuged at 9000 rpm for 1 minute. The tube was rotated in the centrifuge and centrifuged again for another minute.
• Detection with anti-double-stranded DNA antibody After a one-hour incubation at 50°/60°C with shaking, the tube was placed on ice, then centrifuged briefly. The whole volume was dispensed in one microwell of a GEN-ETI-K DEIA coated strip (DiaSorin, code number M2600998143). The strip was covered with a cardboard sealer and incubated overnight at 4°C.
• the strip was washed 5 times with wash buffer ETI-2 (DiaSorin) using an ETY-SYSTEM washer (DiaSorin) or its equivalent.
• Anti-double-stranded DNA antibody 27-14-D9 was diluted 1:50 in anti-double-stranded DNA diluent (DiaSorin, code number M2600999839), and 100 ⁇ l of the diluted antibody was added to the well. After a 30 minute incubation at 37 °C, the well was washed 5 times with ETI-2 buffer.
• the sample was then incubated for 30 minutes at 37 °C with 100 ⁇ l of protein ABS3 enzyme tracer (DiaSorin, code number M2600999876) diluted 1:50 in tracer diluent (DiaSorin, code number M2600999840), and washed 5 times with ETI-2 buffer.
• the sample was incubated for 30 minutes at room temperature in the dark with 100 ⁇ l of a 1:1 mixture of ETI-2 chromogen (DiaSorin, code number M260099803) and ETI-2 substrate (Diasorin, code number M260099804).
• DiaSorin M26000654 blocking reagent 200 ⁇ l was added, and the absorbance of the specimen was measured with a spectrophotometer at 450-630 nm. At least 100 particles of CMV could be detected, as shown in Figure 1.
• This example demonstrates detection of hepatitis C sequences in serum samples using ADDA with the following steps: (a) extraction of total RNA from a serum pellet, (b) conversion of RNA into first strand cDNA using the enzyme reverse transcriptase, (c) liquid-phase hybridization of cDNA with a biotinylated probe, and
• HCV RNA genome was accomplished using the Tripure Isolation Reagent (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions. The final pellet containing RNA was resuspended in 30 ⁇ l of diethyl-pyrocarbonate- treated H 2 0 (SIGMA Chemicals, St. Louis, MO).
• RNA into first strand cDNA by the enzyme reverse transcriptase.
• First strand cDNA synthesis was performed using the reverse transcriptase enzyme Superscript II (Life Technology), using random examers (Life).
• RNA sample 11 ⁇ l
• 1 ⁇ l of examers 250 ng/ ⁇ l
• 5x first strand synthesis buffer 250 mM Tris HCl , pH 8.3, 375 mM KCl, 15 mM MgCl 2
• 2 ⁇ l of 0.2 M DTT 1 ⁇ l of dNTPs (10 mM each)
• l ⁇ l of Superscript a total reaction volume of 20 ⁇ l.
• the mixture was incubated at 42°C for 1 hour. After that time, the enzyme was inactivated by heating the sample to 65°C for 10 minutes.
• the assay was carried out as follows. After capture of double-stranded hybrids, the wells were washed seven times with 300 ⁇ l of washing buffer (6.7 mM phosphate buffer, pH 6.4, 0.13 MNaCl, 0.1% of the detergent "TWEEN 20"). Anti-double stranded DNA antibody 27-14-D9 was added and allowed to react for
• Double-stranded DNA-antibody complexes were detected by adding to the wells a solution containing a horseradish peroxidase-labeled goat-antimouse Ig antibody, incubating for 30 minutes and, after an additional five washes, adding a chromogenic solution (27 g/1 tetramethylbenzidine and 0.1 ml/1 hydrogen peroxide).
• This example demonstrates the use of the AIDA method to detect HTV sequences in the serum of HIV-infected patients.
• Plasma from HIN-infected patients was collected in EDTA or in sodium citrate; heparinized plasma can also be used.
• R ⁇ A was isolated from the serum samples using a QIAamp VIRAL R ⁇ A kit by QIAGEN. The precipitate was redissolved in buffer AVL/carrier RNA by incubation at 80 °C for not more than 5 minutes.
• Prepared buffer AVL (560 ⁇ l) was added to a 1.5 ml centrifuge tube.
• Plasma 140 ⁇ l was added to the tube and the mixture was vortexed and incubated at room temperature 10 minutes. The sample was mixed thoroughly with 560 ⁇ l of ethanol.
• RNA was eluted from the column with 50 ⁇ l of preheated (80 °C) RNase-free water incubated at 80 °C for 5 minutes in a heating block, and centrifuged at 9000 rpm for 1 minute. The tube was rotated 180° in the centrifuge and spun again for another minute.
• cDNA was synthesized using the Superscript TM II RNase H-reverse transcriptase enzyme (Gibco BRL Cat. No. 18064-014). Two microliters of an anti- sense GAG specific primer (250 mg/ ⁇ l) was added to 50 ⁇ l of RNA in Rnase free water.
• the anti-sense GAG specific primer had the following sequence: 5'CTATGTGCCCTTCTTGCCACAAT-3' (SEQ ID NO: 19). This primer matches the sequence of HIV- 1 B perfectly and has not more than 3 mismatches with the other subtypes and 5 mismatches with HIV subtype O. These mismatches should not influence the synthesis of the specific cDNAs, however, we have also verified that for this AIDA application it is possible to utilize random primers that eliminate the eventual problems linked to the presence of different subtypes and quasi species.
• RNA and primer were incubated for 5 minutes at 70 °C and for 10 minutes at room temperature. After a brief centrifugation, 28.5 ⁇ l of the following mix was added to each sample: 16 ⁇ l of 5 X first strand buffer (provided with the enzyme), 8 ⁇ l of 0.1 M DTT (provided with the enzyme), 4 ⁇ l of 10 mM dNTP mix (10 mM each of dATP, dGTP, dCTP and dTTP at neutral pH, and 0.5 ⁇ l of
• 50X Denhard's solution 4 ⁇ l of 0.5 M EDTA, pH 8, and 0.9 ⁇ l of 50X TE were added five microliters of a gag-specific biotinylated sense probe (2 ng/ ⁇ l diluted in IX TE buffer) was added, and the mixture was preheated at 70 °C for 5 minutes.
• the sequence of the probe is 5 otGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCA-3' (SEQ
• This method accurately detected HIV sequences in four infected patients with high viremia and did not detect HTV sequences in two non-infected blood donors.
• This example demonstrates that the sensitivity of AIDA can be further improved by adding mononucleotides and a nucleic acid polymerase capable of producing an extension product from the captured target DNA or cDNA/primer complex.
• This extension product creates new double-stranded DNA that, in turn, provides new target epitopes for recognition by the anti-double-stranded DNA antibody.
• Figure 2 clearly show that the addition of the polymerase elongation step after the hybrid capture increased the sensitivity of the assay by at least two log units.
• Figure 3 shows the results of an AIDA test on the sera of nine blood donors (D1-D9) and with the sera of 9 HIV positive patients. The AIDA test accurately detected HTV sequences in eight of the nine HIV positive patients.
• EXAMPLE 5 Using the method described below we analyzed panels of HTV viremic sera and compared the results with those obtained using reference methods of Roche and Chiron.
• cDNA synthesis, hybridization, and detection were carried out as described in Example 3, except that after the first washings of the microwell, 100 ⁇ l of a solution containing 50 mM Tri, pH 8.8, 15 mM (NH 4 ) 2 SO 4 , 7 mM MgCl 2 , 01 mM EDTA, pH 8, 10 mM ⁇ -mercaptoethanol, 0.02 mg/ml BSA, pH 8.8, 80 ⁇ MNTPs, and 0.2 units of T4 DNA Polymerase (Boehringer Mannheim code 1004794) were added to the well, incubated for 30 minutes at 37 °C and washed 6 times before addition of the anti- double-stranded DNA antibody.
• T4 DNA Polymerase Boehringer Mannheim code 1004794
• Hybridization was carried out as follows. Eighty microliters of cDNA (total cDNA reaction) and 8 ⁇ l of IN NaOH were added to a 0.5 ml tube and mixed carefully by pipetting. The tube was incubated for 5 minutes at 100°C, chilled on ice for 5 minutes, then spun in a minicentrifuge. A pre-mixed solution containing 8 ml of HCl and 4 ml of 1M Tris, pH 7.5, was mixed carefully by pipetting.
• hybridization buffer (6 ⁇ l of 20X SSC, 4.8 ⁇ l of 0.5MEDTA, pH 8.0, 4.8 ⁇ l of 50X Denhardt's solution, 0.9 ⁇ L of 50X TE buffer) was added to each tube. Then, 5 ⁇ l (5 ng) of HCV specific 5' biotinylated probe (CORE-890), pre- warmed at 75 °C for 10 minutes, was added to each tube.
• the probe spans an HCV sequence in the region coding for the core protein. This probe sequence is conserved among HCV subtypes. The sequence of the probe is: 5' GGT CAG ATC GTT GGT GGA GTT TAC TTG TTG CCG CGC AGG G 3' (SEQ ID NO.21).
• Table 3 shows that there was a complete concordance between the data obtaining using PCR and using AIDA.
• This example demonstrates detection of HTV sequences in serum samples using AIDA.
• HTV sequences in serum samples by AIDA involved exactly the same procedures as for HCV, except for the use of a different specific biotinylated probe (JA-Pol).
• This probe has the nucleotide sequence shown in SEQ ID NO: 18.
• Four HIV-positive samples and one HTV-negative sample were analyzed by AIDA, as shown in Table 5.
• EXAMPLE 8 This example demonstrates the detection of human genomic sequences encoding the chemokine receptor CC-CKR5.
• genomic DNA was extracted from whole blood, and the DNA sample was processed for hybridization with the same procedures described above for cDNA hybridization, except for the use of the specific biotinylated probe, F12, having the nucleotide sequence shown in SEQ ID NO: 17.
• This example demonstrates detection of hepatitis C virus (HCV) in serum samples using the LEDIA method.
• HCV is a positive-strand RNA virus
• its RNA genome must be preliminarily extracted from a biological sample such as serum and converted into DNA before any process involving a DNA-dependent DNA polymerase can be performed.
• linear enrichment of the HCV genome for detection involves the following steps: (a) extraction of total RNA from serum, (b) conversion of RNA into first strand cDNA by the enzyme reverse transcriptase, (c) specific linear enrichment of cDNA sequences derived from the target RNA, and (d) detection of the enriched single-stranded HCV sequence. (a) Extraction of total RNA from serum.
• HCV RNA genome was extracted from 100 ⁇ l of serum using the Tripure Isolation Reagent (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions.
• the final pellet containing RNA was resuspended in 30 ⁇ l of diethyl-pyrocarbonate-treated H 2 0 (SIGMA Chemicals, St. Louis, MO).
• RNA RNA was prepared, containing 200 ⁇ M dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 200 ⁇ M dTTP, MgCl 2 2 mM, 16 mMNH 4 (SO 4 ) 2 , 67 mM Tris Cl, pH 8.8, 100 pmoles of a single HCV primer (either HCV A, B, G or 2CH, ), 50 units m of AmpliTaq (Perkin Elmer-Cetus, Norwalk, CT). To this mixture was added 1 ⁇ l of cDNA derived either from HCV positive patients or healthy blood donors negative for any HCV marker.
• a single HCV primer either HCV A, B, G or 2CH,
• AmpliTaq Perkin Elmer-Cetus, Norwalk, CT.
• This mixture was subjected to 35 cycles, each composed of three steps: denaturation at 94°C for one minute, annealing at 50°C for one minute, and primer elongation at 72°C for one minute.
• the cycling reaction was performed using a DNA Thermal Cycler (Perkin Elmer-Cetus, Norwalk, CT).
• the single-stranded enriched sequence was detected using a specific biotinylated probe and an anti-double stranded DNA antibody which is able to discriminate single-stranded from double-stranded DNA, e.g. target DNA hybridized or unhybridized with probe.
• the double-stranded DNA hybrids were visualized by generating a colorimetric signal.
• One fourth of the enrichment reaction was denatured at 100°C for 5 min, then added to streptavidin-coated microtiter wells sensitized with 5 ng of biotinylated ⁇ -3CH probe (SEQ ID NO:7) and hybridized at 55°C for one hour.
• the immunochemical reaction was carried out as described in Example 1, above. Eleven HCV-positive and four HCV-negative serum samples were analyzed by both LEDIA and PCR.
• the PCR reaction for detecting HCV was carried out as follows.
• a reaction mixture was prepared in 100 ⁇ l containing dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 200 ⁇ M dTTP, 2 mM MgCl 2 , 16 mM NH 4 (SO 4 ) 2 , 67 mM Tris Cl , pH 8.8, 50 pmoles of each primer 1CH (SEQ ID NO: 14) and 2CH (SEQ ID NO:4), 50 units/ml of AmpliTaq (Perkin Elmer-Cetus, Norwalk, CT). To this mixture was added 1 ⁇ l of cDNA derived either from HCV positive patients or healthy blood donors, negative for any HCV marker.
• the PCR reaction was carried out for 45 cycles, each composed of three steps: a denaturation step at 94°C for 1 minute, an annealing step at 50°C for 1 minute, and an elongation step at 72°C for 1 minute. At the end of the reaction, an aliquot of 25 ⁇ l was analyzed using agarose gel electrophoresis. A band of amplified DNA measuring 299 base pairs was detected.
• Table 7 compares the results of the LEDIA method with the PCR assay.
• This example demonstrates the detection of hepatitis G viral sequences using the LEDIA method.
• a 100 ⁇ l reaction mixture was prepared containing dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 200 ⁇ M dTTP, MgCl 2 2 mM, 16 mM NH 4 (SO 4 ) 2 , 67 mM Tris HCl, pH 8.8, 50 pmoles of each primer AC1-S (SEQ ID NO: 12) and AC3-AS (SEQ ID NO: 16), 50 units/ml of AmpliTaq (Perkin Elmer-Cetus, Norwalk, CT).
• LEDIA is capable of distinguishing hepatitis G-positive and hepatitis G- negative serum samples.
• This example demonstrates detection of hepatitis B sequences in serum samples using LEDIA.
• Hepatitis B DNA was extracted from 1 ml serum samples as follows. One milliliter of serum was digested for one hour at 56°C with 1 mg/ml proteinase K , in a total volume of 2 ml containing 0.01 M Tris Cl, pH 7.5, 0.01 M EDTA, 150 mM NaCl and 0.5 % sodium-dodecyl-sulfate (SDS). The sample was then extracted using one volume of a 1 : 1 solution of phenol-chloroform (BDH Laboratory Supplies,
• the linear enrichment reaction was carried out as described in Example 4, above, using 40 cycles of enrichment.
• a serum sample containing a known amount of viral genomes (10 7 genomes/ml) was used as a positive control.
• HCV primers SEQ ID NOS: 1-5) and one HBV primer (SEQ ID NO:8) were employed, each at different final concentration and at different temperatures. The relative contribution of each primer and of each primer concentration to the enrichment of single-stranded DNA analyte was studied.
• HCV cDNA was tested by LEDIA using three thermostable DNA polymerase preparations (AmpliTAq and GoldTaq Polymerase from Perkin Elmer-Cetus, and Pwo DNA polymerase from

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