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EP0717782A1 - Amplification dependant de la liaison s'appliquant a la detection d'agents pathogenes infectieux et de genes anormaux - Google Patents

Amplification dependant de la liaison s'appliquant a la detection d'agents pathogenes infectieux et de genes anormaux

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Publication number
EP0717782A1
EP0717782A1 EP95923925A EP95923925A EP0717782A1 EP 0717782 A1 EP0717782 A1 EP 0717782A1 EP 95923925 A EP95923925 A EP 95923925A EP 95923925 A EP95923925 A EP 95923925A EP 0717782 A1 EP0717782 A1 EP 0717782A1
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EP
European Patent Office
Prior art keywords
amplification
nucleic acid
probe
target nucleic
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95923925A
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German (de)
English (en)
Inventor
David Y. Zhang
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Icahn School of Medicine at Mount Sinai
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Mount Sinai School of Medicine
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Publication date
Application filed by Mount Sinai School of Medicine filed Critical Mount Sinai School of Medicine
Publication of EP0717782A1 publication Critical patent/EP0717782A1/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]

Definitions

  • the present invention relates to assays and kits for carrying out said assays for the rapid, automated detection of infectious pathogenic agents and normal and abnormal genes.
  • PCR poly erase chain reaction
  • LCR ligase chain reaction
  • One of the most difficult problems is preparation of the target nucleic acid prior to carrying out its amplification and detection. This process is time and labor intensive and, thus, generally unsuitable for a clinical setting, where rapid and accurate results are required.
  • Another problem, especially for PCR and SDA, is that conditions for amplifying the target nucleic acid for subsequent detection and optional quantitation vary with each test, i.e. , there are no constant conditions favoring test standardization. This latter problem is especially critical for the quantitation of a target nucleic acid by competitive PCR and for the simultaneous detection of multiple target nucleic acids.
  • Circumvention of the aforementioned problems would allow for development of rapid standardized assays, utilizing the various techniques mentioned above, that would be particularly useful in performing epidemiologic investigations, as well as in the clinical laboratory setting for detecting pathogenic microorganisms and viruses in a patient sample.
  • microorganisms cause infectious diseases that represent a major threat to human health.
  • the development of standardized and automated analytical techniques and kits therefor, based on rapid nd sensitive identification of target nucleic acids specific for an infectious disease agent would provide advantages over techniques involving immunologic or culture detection of bacteria and viruses.
  • Reagents may be designed to be specific for a particular organism or for a range of related organisms. These reagents could be utilized to directly assay microbial genes conferring resistance to various antibiotics and virulence factors resulting in disease. Development of rapid standardized analytical techniques will aid in the selection of the proper treatment.
  • assays having a moderate degree of sensitivity may suffice, e.g.. in initial screening tests.
  • great sensitivity is required, e.g. , the detection of the HIV genome in infected blood may require finding the virus nucleic acid sequences present in a sample of one part per 10 to 100,000 human genome equivalents (Harper et al.. Proc. Nat'l. Acad. Sci., USA 82:772, 1986).
  • HIV infectious virus
  • HTLV-I hepatitis B
  • hepatitis C hepatitis C
  • the HIV genome can be detected in a blood sample using PCR techniques, either as an RNA molecule representing the free viral particle or as a DNA molecule representing the integrated provirus (Ou et al. Science 239:295. 1988; Murakawa et al.. DNA 2:287, 1988) .
  • Such techniques include detection and characterization of single gene genetic disorders in individuals and in populations (see, e.g. , Landergren et al.. Science 241: 1077, 1988 which discloses a ligation technique for detecting single gene defects, including point mutations) .
  • Such techniques should be capable of clearly distinguishing single nucleotide differences (point mutations) that can result in disease (e.g. , sickle cell anemia) as well as deleted or duplicated genetic sequences (e.g. , thalassemia) .
  • the methods referred to above are relatively complex procedures that, as noted, suffer from drawbacks making them difficult to use in the clinical diagnostic laboratory for routine diagnosis and epidemiological studies of infectious diseases and genetic abnormalities. All of the methods described involve amplification of the target nucleic acid to be detected. The extensive time and labor required for target nucleic acid preparation, as well as variability in amplification templates (e.g.. the specific target nucleic acid whose detection is being measured) and conditions, render such procedures unsuitable for standardization and automation required in a clinical laboratory setting.
  • the present invention is directed to the development of rapid, sensitive assays useful for the detection and monitoring of pathogenic organisms, as well as the detection of abnormal genes in an individual. Moreover, the methodology of the present invention can be readily standardized and automated for use in the clinical laboratory setting.
  • the improved methodology also allows for rapid and sensitive detection and quantitation of genetic variations in nucleic acids in samples from patients with genetic diseases or neoplasia.
  • This method provides several advantages over prior art methods.
  • the method simplifies the target nucleic acid isolation procedure, which can be performed in microtubes, microchips or micro-well plates, if desired.
  • the method allows for isolation, amplification and detection of nucleic acid sequences corresponding to the target nucleic acid of interest to be carried out in the same sample receptacle, e.g. , tube or micro-well plate.
  • the method allows for standardization of conditions, because, only a pair of generic amplification probes may be utilized in the present method for detecting a variety of target nucleic acids.
  • the amplification probes which in the method may be covalently joined end to end, form a contiguous ligated amplification sequence.
  • This ligated amplification sequence rather than the target nucleic acid, is either directly detected or amplified. allowing for substantially the same amplification conditions to be used for a variety of different infectious agents and, thus, leading to more controlled and consistent results being obtained.
  • multiple infectious agents in a single sample may be detected using the methodology disclosed.
  • Additional advantages of the present invention include the ability to automate the protocol of the method disclosed, which is important in performing routine assays, especially in the clinical laboratory and the ability of the method to utilize various nucleic acid amplification systems, e.g.. poly erase chain reaction (PCR) , strand displacement amplification (SDA) , ligase chain reaction (LCR) , self-sustained sequence replication (3SR) and Q ⁇ replicase amplification (Q ⁇ RA) .
  • PCR poly erase chain reaction
  • SDA strand displacement amplification
  • LCR ligase chain reaction
  • 3SR self-sustained sequence replication
  • Q ⁇ RA Q ⁇ replicase amplification
  • the present method incorporates magnetic separation techniques using paramagnetic particles or beads coated with a ligand binding moiety that recognizes and binds to a ligand on an oligonucleotide capture probe to isolate a target nucleic acid (DNA or RNA) from a sample of a clinical specimen containing e.g.. a suspected pathogenic microorganism or gene abnormality, in order to facilitate detection of the underlying disease-causing agent.
  • DNA or RNA target nucleic acid
  • a target nucleic acid is hybridized to a pair of non-overlapping oligonucleotide amplification probes in the presence of paramagnetic beads coated with a ligand binding moiety, e.g.. streptavidin, to form a complex.
  • a ligand binding moiety e.g.. streptavidin
  • the capture/amplification probe contains a ligand, e.g.. biotin, that is recognized by and binds to the ligand binding moiety on the paramagnetic beads.
  • the probes are designed so that each contains generic sequences (i.e..
  • the two probes may be are joined together using a ligating agent to form a continguous ligated amplification sequence.
  • the ligating agent may be an enzyme, e.g.. DNA ligase or a chemical.
  • the detection of the target nucleic acid in the original sample is determined by detection of the ligated amplification sequence.
  • the ligated amplification sequence may be directly detected if a sufficient amount (e.g...
  • the ligated amplification sequence (not the target nucleic acid) may be amplified using suitable amplification techniques, e.g. PCR, for detection. Alternatively, capture and amplification functions may be performed by separate and independent probes. Unligated probes, as well as the target nucleic acid, are not amplified in this technique.
  • Fig. 1 is a generic schematic diagram showing the various components used in the present method of capture, ligation-dependent amplification and detection of a target nucleic acid.
  • Fig. 2 is a schematic flow diagram generally showing the various steps in the present method.
  • Fig. 3 is an autoradiograph depicting the detection of a PCR amplified probe that detects HIV-1 RNA.
  • Lane A is the ligated amplification sequence according to the invention;
  • Lane B which is a control, is PCR amplified nanovariant DNA, that does not contain any HIV-1-specific sequences.
  • Fig. 4 is a schematic diagram of an embodiment of the present invention showing the various components used for capture and ligation-dependent detection of a target nucleic acid, e.g. HCV RNA, and subsequent amplification of its sequences, employing two capture/amplification probes containing a bound biotin moiety and two ligation-dependent amplification probes.
  • Fig. 5 is a schematic flow diagram showing magnetic isolation, target specific ligation and PCR amplification for the detection of HCV RNA using a single capture/amplification probe and two amplification probes.
  • Fig. 6 is a schematic diagram showing the various components used to amplify and detect a target nucleic acid e.g. HCV RNA, employing two capture/amplification probes, each containing a bound biotin moiety, and a single amplification probe.
  • a target nucleic acid e.g. HCV RNA
  • Fig. 7 is a schematic diagram showing various components used to detect a target nucleic acid e.g. HCV RNA, employing two capture/amplification probes, each containing a bound biotin moiety, and a single amplification probe that circularizes upon hybridization to the target nucleic acid and ligation of free termini.
  • a target nucleic acid e.g. HCV RNA
  • Fig. 8 is a photograph of ethidiu bromide stained DNA depicting PCR amplified probes used to detect HCV RNA in a sample. The amount of HCV RNA in the sample is determined by comparing sample band densities to those of standard serial dilutions of HCV transcripts.
  • Fig. 9. is a photograph of ethidium bromide stained DNA depicting PCR amplified single, full length ligation-dependent and circularizable probes used to detect HCV RNA in a sample. The amount of HCV RNA in the sample is determined by comparing sample band densities to those of standard serial dilutions of HCV transcripts.
  • the present invention is directed towards simplified sample preparation and generic amplification systems for use in clinical assays to detect and monitor pathogenic microorganisms in a test sample, as well as to detect abnormal genes in an individual.
  • Generic amplification systems are described for clinical use that combine magnetic separation techniques with ligation/amplification techniques for detecting and measuring nucleic acids in a sample.
  • the separation techniques may be combined with most amplification systems, including inter alia. PCR, LCR, SDA and Q ⁇ amplification techniques.
  • the advantages of the present invention include (1) suitability for clinical laboratory settings, (2) ability to obtain controlled and consistent (standardizable) results, (3) ability to quantitate nucleic acids in a particular sample and (4) ability to simultaneously detect and quantitate multiple target nucleic acids in a test sample.
  • the complete protocol of the presently disclosed method may be easily automated, making it useful for routine diagnostic testing in a clinical laboratory setting.
  • the present invention incorporates magnetic separation, utilizing paramagnetic particles, beads or spheres that have been coated with a ligand binding moiety that recognizes and binds to ligand present on an oligonucleotide capture probe, described below, to isolate a target nucleic acid (DNA or RNA) from a clinical sample in order to facilitate its detection.
  • Magnetic separation is a system that uses paramagnetic particles or beads coated with a ligand binding moiety to isolate a target nucleic acid (RNA or DNA) (Lomeli et al. Clin. Chem. 3_5:1826, 1989) from a sample.
  • the principle underscoring this method is one of hybrid formation between a capture probe containing a ligand, and a target nucleic acid through the specific complementary sequence between the probe and target.
  • Hybridization is carried out in the presence of a suitable chaotropic agent, e.g.. guanidine thiocyanate (GnSCN) which facilitates the specific binding of the probe to complementary sequences in the target nucleic acid.
  • GnSCN guanidine thiocyanate
  • ligand refers to any component that has an affinity for another component termed here as "ligand binding moiety.”
  • the binding of the ligand to the ligand binding moiety forms an affinity pair between the two components.
  • affinity pairs include, inter alia, biotin with avidin/streptavidin, antigens or haptens with antibodies, heavy metal derivatives with thiogroups, various polynucleotides such as homopolynucleotides as poly dG with poly dC, poly dA with poly dT and poly dA with poly U.
  • Any component pairs with strong affinity for each other can be used as the affinity pair, ligand-ligand binding moiety.
  • Suitable affinity pairs are also found among ligands and conjugates used in immunological methods.
  • the preferred ligand-ligand binding moiety for use in the present invention is the biotin/streptavidin affinity pair.
  • the present invention provides for the capture and detection of a target nucleic acid as depicted in Fig. 1, which provides a schematic depiction of the capture and detection of a target nucleic acid.
  • Fig. 1 provides a schematic depiction of the capture and detection of a target nucleic acid.
  • the target nucleic acid is hybridized simultaneously to a pair of oligonucleotide amplification probes, i.e.. a first nucleotide probe (also referred to as a capture/amplification probe) and a second nucleotide probe (also referred to as an amplification probe) , designated in Fig.
  • the probes may be either oligodeoxyribonucleotide or oligoribonucleotide molecules, with the choice of molecule type depending on the subsequent amplification method, e.g.. PCR (deoxyribonucleotides) or Q ⁇ replicase (either deoxyribo- or ribonucleotides) to be used.
  • PCR deoxyribonucleotides
  • Q ⁇ replicase either deoxyribo- or ribonucleotides
  • the capture/amplification probe is designed to have a generic 3' nucleotide sequence (d) , i.e.. it is not specific for the specific target nucleic acid being analyzed and thus can be used with a variety of target nucleic acids.
  • the 3' sequence of the first probe is not complementary, nor hybridizable, to the nucleotide sequence of the target nucleic acid.
  • the 5' portion (e) of the capture/amplification probe comprises a nucleotide sequence that is complementary and hybridizable to a portion of the nucleotide sequence of the specific target nucleic acid.
  • the capture/amplification probe is synthesized so that its 3' generic sequence (d) is the same for all systems, with the 5' specific sequence (e) being specifically complementary to a target nucleic acid of an individual species or subspecies of organism or an abnormal gene, e.g. the gene(s) responsible for cystic fibrosis or sickle cell anemia.
  • the 5' specific portion of the capture/amplification probe be specifically complementary to the nucleotide sequence of a target nucleic acid of a particular strain of organism.
  • Capture/Amp-probe-1 further contains a ligand (c) at the 3' end of the probe (d) , which is recognized by and binds to the ligand binding moiety (b) coated onto the paramagnetic beads (a) .
  • the second or amplification probe i.e. , Amp- probe-2 in Fig. 1, contains a 3' sequence (f) that is complementary and hybridizes to a portion of the nucleotide sequence of a target nucleic acid immediately adjacent to (but not overlapping) the sequence of the target that hybridizes to the 5' end of Capture/Amp-probe-1.
  • Amp-probe-2 also contains a 5' generic sequence (g) which is neither complementary nor hybridizable to the target nucleic acid, to which may be optionally attached at the 5' end thereof a label or signal generating moiety (***) .
  • signal generating moieties include, inter alia, radioisotopes, e.g.
  • fluorescent molecules e.g.. fluorescein and chromogenic molecules or enzymes, e.g.. peroxidase.
  • fluorescent molecules e.g.. fluorescein and chromogenic molecules or enzymes, e.g.. peroxidase.
  • Such labels are used for direct detection of the target nucleic acid and detects the presence of Amp-probe-2 bound to the target nucleic acid during the detection step.
  • 32 P is preferred for detection analysis by radioisotope counting or autoradiography of electrophoretic gels.
  • Chromogenic agents are preferred for detection analysis, e.g.. by an enzyme linked chromogenic assay.
  • target nucleic acid hybridized to the specific 5' portion of the probe is captured by the paramagnetic beads.
  • Amp- probe-2 which has also hybridized to the target nucleic acid is also captured by the paramagnetic beads.
  • the probes are ligated together (at the site depicted by the verticle arrow in Fig. 1) using a ligating agent to form a contiguous single-stranded oligonucleotide molecule, referred to herein as a ligated amplification sequence.
  • the ligating agent may be an enzyme, e.g.. a DNA or RNA ligase, or a chemical joining agent, e.g.. cyanogen bromide or a carbodii ide (Sokolova et al.. FEBS Lett. 131:153-155, 1988).
  • the ligated amplification sequence is hybridized to the target nucleic acid (either an RNA or DNA) at the region of the ligated amplification sequence that is complementary to the target nucleic acid (e.g.. (e) and (f) in Fig. 1) .
  • target nucleic acid e.g. 10 6 - 10 7 molecules
  • detection of the target nucleic acid can be achieved without any further amplification of the ligated amplification sequence, e.g.. by detecting the presence of the optional signal generating moiety of at the 5' end of Amp-probe-2.
  • target nucleic acid e.g. , ⁇ 10 6 molecules
  • the ligated amplification sequence formed as described above by the ligation of Capture/Amp-probe-1 and Amp-probe-2 may be amplified for detection as described below.
  • the present method may be used with routine clinical samples obtained for testing purposes by a clinical diagnostic laboratory.
  • Clinical samples that may be used in the present method include, inter alia, whole blood, separated white blood cells, sputum, urine, tissue biopsies, throat swabbings and the like, any patient sample normally sent to a clinical laboratory for analysis.
  • Fig. 2 provides a general diagramatic description of the magnetic separation and target-dependent detection of a target nucleic acid in a sample
  • this aspect of the present method involves the following steps:
  • the first step is the capture or isolation of a target nucleic acid present in the sample being analyzed, e.g.. serum.
  • a suitable sample size for analysis that lends itself well to being performed in a micro-well plate is about lOO ⁇ l.
  • the use of micro-well plates for analysis of samples by the present method facilitates automation of the method.
  • the sample, containing a suspected pathogenic microorganism or virus or abnormal gene is incubated with an equal volume of lysis buffer, containing a chaotropic agent (i.e.. an agent that disrupts hydrogen bonds in a compound) , a stabilizer and a detergent, which provides for the release of any nucleic acids and proteins that are present in the sample.
  • a chaotropic agent i.e. an agent that disrupts hydrogen bonds in a compound
  • a suitable lysis buffer for use in the present method comprises 2.5 - 5M guanidine thiocyanate (GnSCN) , 10% dextran sulfate, lOOmM EDTA, 200mM Tris-HCl(pH 8.0) and 0.5% NP-40 (Nonidet P-40, a nonionic detergent, N- lauroylsarcosine, Sigma Chemical Co., St. Louis, MO).
  • GnSCN guanidine thiocyanate
  • NP-40 Nonidet P-40, a nonionic detergent, N- lauroylsarcosine, Sigma Chemical Co., St. Louis, MO.
  • the concentration of GnSCN, which is a chaotropic agent, in the buffer also has the effect of denaturing proteins and other molecules involved in pathogenicity of the microorganism or virus. This aids in preventing the possibility of any accidental infection that may occur during subsequent manipulations of samples containing pathogens.
  • Paramagnetic particles or beads coated with the ligand binding moiety are added to the sample, either simultaneous with or prior to treatment with the lysis buffer.
  • the paramagnetic beads or particles used in the present method comprise ferricoxide particles (generally ⁇ 1 urn in diameter) that possess highly convoluted surfaces coated with silicon hydrides.
  • the ligand binding moiety is covalently linked to the silicon hydrides.
  • the paramagnetic particles or beads are not magnetic themselves and do not aggregate together. However, when placed in a magnetic field, they are attracted to the magnetic source. Accordingly, the paramagnetic particles or beads, together with anything bound to them, may be separated from other components of a mixture by placing the reaction vessel in the presence of a strong magnetic field provided by a magnetic separation device.
  • Such devices are commercially available, e.g.. from Promega Corporation or Stratagene, Inc.
  • Suitable paramagnetic beads for use in the present method are those coated with streptavidin, which binds to biotin.
  • streptavidin which binds to biotin.
  • Such beads are commercially available from several sources, e.g. , Streptavidin MagneSphere® paramagnetic particles obtainable from Promega Corporation and Streptavidin-Magnetic Beads (catalog #MB002) obtainable from American Qualex, La Mirada, CA.
  • a pair of oligonucleotide amplification probes is added to the lysed sample and paramagnetic beads.
  • the probes and paramagnetic beads may be added at the same time.
  • the two oligonucleotide probes are a first probe or capture/amplification probe (designated Capture/Amp- probe-1 in Fig. 1) containing a ligand at its 3' end and a second probe or amplification probe (designated Amp-probe-2 in Fig. 1) .
  • the first probe is preferably a 3'-biotinylated capture/amplification probe.
  • the probes may be synthesized from nucleoside triphosphates by known automated oligonucleotide synthetic techniques, e.g.. via standard phosphoramidite technology utilizing a nucleic acid synthesizer. Such synthesizers are available, e.g.. from Applied Biosystems, Inc. (Foster City, CA) .
  • Each of the oligonucleotide probes are about 40- 200 nucleotides in length, preferably about 50-100 nucleotides in length, which, after ligation of the probes, provides a ligated amplification sequence of about 80-400, preferably 100-200, nucleotides in length, which is suitable for amplification via PCR, Q ⁇ replicase or SDA reactions.
  • the target nucleic acid specific portions of the probes i.e. , the 5' end of the first capture/amplification probe and the 3' end of the second amplification probe complementary to the nucleotide sequence of the target nucleic acid, are each approximately 15-60 nucleotides in length, preferably about 18-35 nucleotides, which provides a sufficient length for adequate hybridization of the probes to the target nucleic acid.
  • the generic nucleotide sequence of an oligodeoxynucleotide capture/amplification probe comprises at least one and, preferably two to four, restriction endonuclease recognition sequences(s) of about six nucleotides in length, which can be utilized, if desired, to cleave the ligated amplification sequence from the paramagnetic beads by specific restriction endonucleases, as discussed below.
  • Preferred restriction sites include, inter alia. .EcoRI (GAATTC) , Smal (CCCGGG) and HindiII (AAGCTT) .
  • the generic nucleotide sequence comprises a G-C rich region which, upon hybridization to a primer, as discussed below, provides a more stable duplex molecule, e.g.. one which requires a higher temperature to denature.
  • Ligated amplification sequences having G- C rich generic portions of the capture/amplification and amplification probes may be amplified using a two temperature PCR reaction, wherein primer hybridization and extension may both be carried out at a temperature of about 60-65°C (as opposed to hybridizing at 37°C, normally used for PCR amplification) and denaturation at a temperature of about 92°C, as discussed below.
  • the use of a two temperature reaction reduces the length of each PCR amplification cycle and results in a shorter assay time.
  • a ternary complex comprising the target nucleic acid and hybridized probes is formed, which is bound to the paramagnetic beads through the binding of the ligand (e.g.. biotin) on the capture/amplification probe to the ligand binding moiety (e.g.. streptavidin) on the paramagnetic beads.
  • the ligand e.g.. biotin
  • the ligand binding moiety e.g.. streptavidin
  • the complex comprising target nucleic acid-probes-beads is then separated from the lysis buffer by means of a magnetic field generated by a magnetic device, which attracts the beads.
  • the magnetic field is used to hold the complex to the walls of the reaction vessel, e.g. , a micro-well plate or a microtube, thereby allowing for the lysis buffer and any unbound reactants to be removed, e.g. , by decanting, without any appreciable loss of target nucleic acid or hybridized probes.
  • the complex is then washed 2-3 times in the presence of the magnetic field with a buffer that contains a chaotropic agent and detergent in amounts that will not dissociate the complex.
  • a suitable washing buffer for use in the present method comprises about 1.0 - 1.5M GnSCN, lOmM EDTA, lOOmM Tris-HCl (pH 8.0) and 0.5% NP-40 (Nonidet P-40, nonionic detergent, Sigma Chemical Co., St. Louis, MO) .
  • NP-40 Nonidet P-40, nonionic detergent, Sigma Chemical Co., St. Louis, MO
  • Other nonionic detergents e.g. , Triton X- 100, may also be used.
  • the buffer wash removes unbound proteins, nucleic acids and probes that may interfere with subsequent steps.
  • the washed complex may be then washed with a solution of KC1 to remove the GnSCN and detergent and to preserve the complex.
  • a suitable concentration of KC1 is about 100 to 500mM KC1.
  • the KC1 wash step may be omitted in favor of two washes with ligase buffer.
  • the next step in the present method involves treating the complex from step (a) with a ligating agent that will join the two probes.
  • the ligating agent may be an enzyme, e.g.. DNA or RNA ligase, or a chemical agent, e.g.. cyanogen bromide or a carbodiimide. This serves to join the 5' end of the first oligonucleotide probe to the 3' end of the second oligonucleotide probe (capture/amplification probe and amplification probe, respectively) to form a contiguous functional single-stranded oligonucleotide molecule, referred to herein as a ligated amplification sequence.
  • the ligated amplification sequence serves as the template for any of various amplification systems, such as PCR, SDA or Q ⁇ replicase amplification. Any of the first and second probes which remain unligated after treatment are not amplified in subsequent steps in the method.
  • Capture/amplification and amplification oligodeoxynucleotide probes may be ligated using a suitable ligating agent, such as a DNA or RNA ligase.
  • the ligating agent may be a chemical, such as cyanogen bromide or a carbodiimide (Sokolova et al.. FEBS Lett. 231:153-155, 1988).
  • Preferred DNA ligases include T 4 DNA ligase and the thermostable Taq DNA ligase, with the latter being most preferable, for probes being subjected to amplification using PCR techniques.
  • the advantage of using the Taq DNA ligase is that it is active at elevated temperatures (65- 72°C) . Joining the oligonucleotide probes at such elevated temperatures decreases non-specific ligation.
  • the ligation step is carried out for 30-60 minutes at an elevated temperature (about 65-72°C) , after which time any unligated second amplification probe (Amp-probe-2 in Fig. 1) may be, optionally, removed under denaturing conditions.
  • an elevated temperature about 65-72°C
  • Denaturation is performed after the ligation step by adding TE Buffer (lOmM Tris-HCl pH 7.5, O.lmM EDTA) to the mixture. The temperature of the mixture is then raised to about 92-95°C for about 1-5 minutes to denature the hybridized nucleic acid. This treatment separates the target nucleic acid (and unligated Amp- probe-2) from the hybridized ligated amplification sequences, which remains bound to the paramagnetic beads.
  • TE Buffer lauent Tris-HCl pH 7.5, O.lmM EDTA
  • the third step in the process is detection of the ligated amplification sequence, which indicates the presence of the target nucleic acid in the original test sample. This may be performed directly if sufficient target nucleic acid (about 10 6 - 10 7 molecules) is present in the sample or following amplification of the ligated amplification sequence, using one of the various amplification techniques, e.g.. PCR, SDA, or Q ⁇ replicase amplification. For example, direct detection may be used to detect HIV-1 RNA in a serum sample from an acutely infected AIDS patient. Such a serum sample is believed to contain about 10 6 copies of the viral RNA/ l.
  • an oligonucleotide detection probe of approximately 10-15 nucleotides in length, prepared by automative synthesis as described above to be complementary to the 5' end of the Amp-probe-2 portion of the ligated amplification sequence, may be added to the ligated amplification sequence attached to the paramagnetic beads.
  • the detection probe which is labelled with a signal generating moiety, e.g.. a radioisotope, a chromogenic agent or a fluorescent agent, is incubated with the complex for a period of time and under conditions sufficient to allow the detection probe to hybridize to the ligated amplification sequence.
  • the incubation time can range from about 1-60 minutes and may be carried out at a temperature of about 4-60°C.
  • the incubation temperature is about 4°C; a chromogenic agent, about 37°C; and a radioisotope, about 37° - 60°C.
  • Preferred signal generating moieties include, inter alia. 32 P (radioisotope) , peroxidase (chromogenic) and fluorescein, acridine or ethidium (fluorescent) .
  • the Amp-probe-2 itself may be optionally labeled at its 5' end with a signal generating moiety, e.g. f 32 P.
  • the signal generating moiety will then be incorporated into the ligated amplification sequence following ligation of the Capture/Amp-probe-1 and Amp- probe-2.
  • direct detection of the ligated amplification sequence to indicate the presence of the target nucleic acid, can be carried out immediately following ligation and washing.
  • Any suitable technique for detecting the signal generating moiety directly on the ligated amplification probe or hybridized thereto via the detection primer may be utilized.
  • Such techniques include scintillation counting (for 32 P) and chromogenic or fluorogenic detection methods as known in the art.
  • suitable detection methods may be found, inter alia, in Sambrook et al. , Molecular Cloning - A Laboratory Manual, 2d Edit., Cold Spring Harbor Laboratory, 1989, in Methods in Enzymology. Volume 152, Academic Press (1987) or Wu et al.. Recombinant DNA Methodology. Academic Press (1989) .
  • an amplification system is used to amplify the ligated amplification sequence for detection.
  • the Q ⁇ replicase amplification technique may be used to amplify either oligoribonucleotide or oligodeoxyribonucleotide ligated amplification sequences using the protocol generally described in Zhang, D.Y., Q ⁇ Replicase Directed RNA Polymerization. Ph.D. Thesis Dissertation, New York University, 1992.
  • the enzyme Q ⁇ replicase is added directly to the reaction vessel to amplify the ligated amplification sequence (i.e. , the ligated Capture/Amp- probe-1 and Amp-probe-2 in Fig. 1) .
  • Q ⁇ replicase amplification may be generally carried out as follows: Between 10 2 and 10 9 molecules (e.g.. about 0.5 attograms to 0.5 nanograms) of ligated amplification sequence are incubated with 48-50 ⁇ g/ml Q ⁇ replicase (about 7 x 10 10 active enzyme molecules) for 20 to 60 min at 37°C in 10 to 25 ⁇ l of 400 ⁇ M ATP, 400 ⁇ M CTP or 40 ⁇ M [ ⁇ - 32 P]CTP, 400 ⁇ M GTP, 5 to 12mM MgCl 2 , and 90mM Tris-HCl (pH 7.5).
  • Q ⁇ replicase about 7 x 10 10 active enzyme molecules
  • the reactions are terminated by adding 10 to 80 ⁇ l of a stop buffer (20mM EDTA (pH7.5), 120mM NaCl) and placing them in an ice bath. Small aliquots are then removed to determine the yield and homogeneity of the oligoribonucleotide product.
  • a stop buffer (20mM EDTA (pH7.5), 120mM NaCl)
  • Small aliquots are then removed to determine the yield and homogeneity of the oligoribonucleotide product.
  • about 12.5 to 25 ⁇ l reactions are initiated with about 10 2 to 10 8 molecules of ligated amplification sequence.
  • 2 to 4 ⁇ l samples are taken at various times and diluted with 18 to 30 ⁇ l of stop buffer. 8 ⁇ l of diluted sample are precipitated by the addition of 50 ⁇ l of an ice-cold precipitation solution (300mM phosphoric acid, 20mM sodium pyrophosphate, and ImM EDTA) .
  • RNA samples are bound to a nitrocellulose membrane (Bio-Rad Corp. , Richmond, CA) on a dot-blot vacuum filtering manifold (Bio-Rad Corp.) and each is washed three times with 150 ⁇ l of ice-cold precipitation solution to remove unincorporated [ ⁇ - 32 P]CTP.
  • the membrane is then soaked in 150 ml of ice-cold precipitation solution for 10 min.
  • the amount of [ 32 P] RNA present in each sample is determined by measuring the radioactivity of each dot-blot.
  • the size of the RNA products in selected samples is determined by analyzing the remainder of the terminated reaction mixture by electrophoresis through an 8% polyacrylamide gel.
  • PCR methodology can be employed to amplify the ligated amplification sequence, using known techniques (see, e.g.. PCR Technology. H.A. Erlich, ed., Stockton Press, 1989, Sambrook et al.. Molecular Cloning - A Laboratory Manual, 2d Edit., Cold Spring Harbor Laboratory, 1989.
  • primers When using PCR for amplification, two primers are employed, the first of the primers being complementary to the generic 3' end of Capture/Amp-probe-1 region of the ligated amplification sequence and the second primer corresponding in sequence to the generic 5' end of Amp-probe-2 portion of the ligated amplification sequence.
  • These primers like the sequences of the probes to which they bind, are designed to be generic and may be used in all assays, irrespective of the sequence of the target nucleic acid.
  • first primer is designed to anneal to the generic sequence at the 3' end of the ligated amplification sequence and the second primer corresponds in sequence to the generic sequence at the 5' end of the ligated amplification sequence
  • generic primers may be utilized to amplify any ligated amplification sequence.
  • a generic pair of PCR oligonucleotide primers for use in the present method may be synthesized from nucleoside triphosphates by known automated synthetic techniques, as discussed above for synthesis of the oligonucleotide probes.
  • the primers may be 10-60 nucleotides in length.
  • the oligonucleotide primers are about 18-35 nucleotides in length, with lengths of 16-21 nucleotides being most preferred.
  • the pair of primers are designated to be complementary to the generic portions of the first capture/amplification probe and second amplification probe, respectively and thus have high G-C content. It is also preferred that the primers are designed so that they do not have any secondary structure, i.e.. each primer contains no complementary region within itself that could lead to self annealing.
  • each cycle of amplification is carried out as follows: Annealing of the primers to the ligated amplifica- tion sequence is carried out at about 37-50°C; exten ⁇ sion of the primer sequence by Taq polymerase in the presence of nucleoside triphosphates is carried out at about 70-75°C; and the denaturing step to release the extended primer is carried out at about 90-95°C.
  • the annealing and extension steps may both be carried at about 60-65°C, thus reducing the length of each amplification cycle and resulting in a shorter assay time.
  • a suitable three temperature PCR amplification (as provided in Saiki et al. , Science 239:487-491. 1988) may be carried out as follows:
  • PCR Polymerase chain reactions
  • Reactions are incubated at 94°C for 1 minute, about 37 to 55°C for 2 minutes (depending on the identity of the primers) , and about 72°C for 3 minutes and repeated for 30-40, preferably 35, cycles.
  • a 4 ⁇ l- aliquot of each reaction is analyzed by electrophoresis through a 2% agarose gel and the DNA products in the sample are visualized by staining the gel with ethidium-bromide.
  • the two temperature PCR technique differs from the above only in carrying out the annealing/extension steps at a single temperature, e.g.. about 60-65°C for about 5 minutes, rather than at two temperatures.
  • quantitative detection of the target nucleic acid using a competitive PCR assay may also be carried out.
  • a oligodeoxyribonucleotide releasing primer synthesized generally as described above, is added to the paramagnetic bead-bound ligated amplification sequence.
  • the releasing primer may or may not be but, preferably, will be the same as the first PCR primer discussed above.
  • the releasing primer is designed to hybridize to the generic 3' end of the Capture/Amp-probe-1 portion of the ligated amplification sequence, which, as discussed above, comprises a nucleotide sequence recognized by at least one, and preferably two-four, restriction endonucleases to form at least one, and preferably two-four, double- stranded restriction enzyme cleavage site, e.g. f a JScoRI, Smal and/or Hindlll site(s).
  • Capture/Amp-probe-1 be synthesized with at least one, and preferably two to four nucleotide sequences recognized by a restriction enzyme located at the 3' end of the probe. This provides the nucleotide sequences to which the releasing primer binds to form double-stranded restriction enzyme cleavage site(s) .
  • the releasing primer is hybridized to the ligated amplification sequence.
  • At least one restriction enzyme e.g.. EcoRI , Smal and/or Hindlll , is then added to the hybridized primer and ligated amplification sequence.
  • the ligated amplification sequence is released from the beads by cleavage at the restriction enzyme, e.g.. EcoRI site.
  • the ligated amplification sequence is serially diluted and then quantitatively amplified via the DNA Taq polymerase using a suitable PCR amplification technique, as described above.
  • Quantitation of the original target nucleic acid in the sample may be performed by a competitive PCR method to quantitatively amplify the ligated amplification sequence, as provided, e.g.. in Sambrook et al.. Molecular Cloning - A Laboratory Manual. 2d Edit., Cold Spring Harbor Laboratory, 1989.
  • the method involves coamplification of two templates: the ligated amplification sequence and a control (e.g.. the generic portions of the ligated amplification sequence or the generic portions that have interposed thereto a nucleotide sequence unrelated to the sequence of the target nucleic acid) added in known amounts to a series of amplification reactions. While the control and ligated amplification sequence are amplified by the same pair of generic PCR primers, the control template is distinguishable from the ligated amplification sequence, e.g.. by being different in size.
  • control and ligated amplification sequence templates are present in the same amplification reaction and use the same primers, the effect of a number of variables which can effect the efficiency of the amplification reaction is essentially nullified.
  • variables included, inter alia. (1) quality and concentration of reagents (Taq DNA polymerase, primers, templates, dNTP's) , (2) conditions used for denaturation, annealing and primer extension, (3) rate of change of reaction temperature and (4) priming efficiency of the oligonucleotide primers.
  • the relative amounts of the two amplified products i.e.. ligated amplification sequence and control template — reflect the relative concentrations of the starting templates.
  • the quantitative PCR method may be generally carried out as follows: 1.
  • a control template e.g.. a DNA sequence corresponding to nanovariant RNA, a naturally occurring template of Q ⁇ replicase (Schaffner et al. , J. Mol. Biol. 117:877-907. 1977) is synthesized by automated oligonucleotide synthesis and its concentration determined, e.g. , by spectrophotometry or by ethidium- bromide mediated fluorescence.
  • a series of tenfold dilutions (in TE Buffer) containing from 10 ng/ml to l fg/ml of the control template is made and stored at -70°C until use.
  • a series of PCR amplification reactions of the free ligated amplification sequence is set up. In addition to the usual PCR ingredients, the reactions also contain about lO ⁇ l/reaction of the tenfold dilutions of the control template and about 10 ⁇ Ci/reaction of [ ⁇ - 32 P] dCTP(Sp.act. 3000 Ci/mmole) .
  • PCR amplification reactions are carried out for a desired number of cycles, e.g.. 30-40.
  • the reaction products may then be subject to agarose gel electrophoresis and autoradiography to separate the two amplified products (of different sizes) .
  • the amplified bands of the control and ligated amplification sequence are recovered from the gel using suitable techniques and radioactivity present in each band is determined by counting in a liquid scintillation counter.
  • the relative amounts of the two products are calculated based on the amount of radioactivity in each band.
  • the amount of radioactivity in the two samples must be corrected for the differences in molecular weights of the two products.
  • the reactions may be repeated using a narrower range of concentration of control template to better estimate the concentration of ligated amplification sequence. In another aspect of the invention, more than the two probes i.e.
  • a single capture/amplification probe, and a single amplification probe may be utilized.
  • one or more capture/amplification probes, and one or more amplification probes may be employed in the detection and capture of the target nucleic acid, and optional amplification of the target sequences, as shown schematically in Figs. 4 and 5.
  • the capture/amplification probes may have a 3' sequence complementary to the target nucleic acid and a biotin moiety at the 5' terminus that is capable of interacting with the streptavidin coated paramagnetic beads.
  • the capture/amplification probes may have a 5' sequence complementary to the target nucleic acid and a biotin moiety at the 3' terminus.
  • one or more amplification probes are utilized such that each probe contains sequences that are specifically complementary to and hybridizable with the target nucleic acid.
  • the 5' end of one amplification probe e.g. Amp-probe-2 (HCV A) in Fig. 4 contains a sequence complementary to a distinct portion in the target nucleic acid.
  • the 3' end of the second amplification probe e.g. Amp-probe-2A (HCV A) in Fig. 4, contains a specific sequence complementary to a region of the target nucleic acid that is immediately adjacent to that portion of the target hybridizable to the first amplification probe.
  • the capture/amplification probes and the pair of amplification probes hybridize with the target nucleic acid in the presence of GnSCN as described above.
  • This complex so formed is bound to streptavidin-coated paramagnetic beads by means of a biotin moiety on the capture/amplification probes and the complex separated from unreacted components by means of magnetic separation as above.
  • the amplification probes may be linked, for example, by a ligase enzyme. This produces a ligated amplification sequence that serves as a template for Taq DNA polymerase during amplification reaction by PCR.
  • two or more capture/amplification probes and two pairs of amplification probes are utilized for the detection of the target nucleic acid.
  • a capture/amplification probe for use in the present method may be designed to bind to the poly-A tail region of cellular mRNA, whereby all such mRNA can be isolated by a single capture-and-wash step.
  • Subsequent PCR amplification may be designed to detect and amplify specific target pathogen or disease gene sequences from such an mRNA pool.
  • genes may include, inter alia, the gene encoding the cystic fibrosis transmembrane regulator protein (CFTR) or hemoglobins or other proteins involved in genetic diseases.
  • CFTR cystic fibrosis transmembrane regulator protein
  • the multiple capture/amplification probes may target, for example, all strains of a particular pathogen, e.g. the Hepatitis C Virus (HCV) , and amplification probes may be tailored to detect and further identify individual HCV genotypes of the pathogen (e.g. HCV) .
  • HCV Hepatitis C Virus
  • two capture/amplification probes are utilized, e.g. as depicted in Fig. 4. This provides a total specific sequence of the capture/amplification probes complementary and hybridizable to the target nucleic acid that can be twice as long as that of a single capture/amplification probe, thereby affording an even higher capture efficiency.
  • the pair of capture/amplification probes may each have a 3' sequence complementary to the target nucleic acid, and a biotin moiety at its 5' terminus capable of interacting with streptavidin coated paramagnetic beads.
  • the pair of capture/amplification probes may each have a 5' sequence complementary to the target nucleic acid, and a biotin moiety at its 3' terminus capable of interacting with streptavidin coated paramagnetic beads.
  • the present invention may utilize a single amplifiable "full length probe” and one or more capture/amplification probes as shown in Fig. 6.
  • the hybridized nucleic acid duplex comprising of the target nucleic acid, for example, HCV RNA
  • the capture/amplification probes and full length amplification probes also referred to as amplification sequences
  • the hybridized duplex comprising of the target nucleic acid, e.g.
  • DNA, and the capture/amplification probes and full length amplification probes can be released from the magnetic beads by treating the hybridized duplex molecule with appropriate restriction enzymes, as described above.
  • the probe When a full length amplification probe is employed to detect a target nucleic acid sequence, the probe may be utilized in amplification reactions such as PCR, without having to use the ligation step described above. This latter approach, in particular, simplifies the assay and is especially useful when at least 10 4 target nucleic acid molecules are available in the testing sample, so that the chances of non-specific binding in a ligation independent detection reaction are reduced.
  • the target nucleic acid such as a pathogen
  • the target nucleic acid is present at >10 5 molecules/ml. of sample, and thus would be amenable to detection and amplification by this method.
  • a still further aspect of the present invention utilizes one or more capture/amplification probes, each containing a biotin moiety, and a single amplification probe, also referred to as an amplification sequence, that hybridizes to the target nucleic acid and circularizes upon ligation of its free termini, as shown in Fig. 7.
  • the amplification probe may be designed so that complementary regions (see e.g. the region shown in bold in Fig.7) of the probe that are hybridizable to the target nucleic acid sequence are located at each end of the probe (as described in Nilsson et al., 1994, Science 165:2085-2088).
  • a linking agent such as a ligase enzyme.
  • This circular molecule may then serve as a template during an amplification step, e.g. PCR, using primers such as those depicted in Fig. 7.
  • the probe can be used to detect different genotypes of a pathogen, e.g. different genotypes of HCV from serum specimens.
  • Genotype specific probes can be designed, based on published HCV sequences (Stuyver et al., 1993, J. Gen. Virol. 7_4: 1093-1102) , such that a mutation in the target nucleic acid is detectable since such a mutation would interfere with (1) proper hybridization of the probe to the target nucleic acid and (2) subsequent ligation of the probe into a circular molecule. Because of the nature of the circularized probe, as discussed below, unligated probes may be removed under stringent washing conditions.
  • the single, full length, ligation-dependent circularizable probe affords greater efficiency of the detection and amplification of the target nucleic acid sequence. Due to the helical nature of double-stranded nucleic acid molecules, circularized probes are wound around the target nucleic acid strand. As a result of the ligation step, the probe may be covalently bound to the target molecule by means of catenation. This results in immobilization of the probe on the target molecule, forming a hybrid molecule that is substantially resistant to stringent washing conditions. This results in significant reduction of non-specific signals during the assay, lower background noise and an increase in the specificity of the assay.
  • the amplification probes and/or amplification sequences as described above can be used for in situ LD-PCR assays.
  • In situ PCR may be utilized for the direct localization and visualization of target viral nucleic acids and may be further useful in correlating viral infection with histopathological finding.
  • Current methods assaying for target viral RNA sequences have utilized RT PCR techniques for this purpose (Nuovo et al., 1993, Am. J. Surg. Pathol. 17(7): 683-690).
  • cDNA obtained from target viral RNA by in situ reverse transcription, is amplified by the PCR method.
  • Subsequent intracellular localization of the amplified cDNA can be accomplished by in situ hybridization of the amplified cDNA with a labelled probe or by the incorporation of labelled nucleotide into the DNA during the amplification reaction.
  • the RT PCR method suffers drawbacks which are overcome by the present invention.
  • various tissue fixatives used to treat sample tissues effect the crosslinking of cellular nucleic acids and proteins, e.g. protein-RNA and RNA-RNA complexes and hinder reverse transcription, a key step in RT-PCR.
  • target RNA may also interfere with reverse transcription.
  • the application of multiplex PCR to RT PCR for the detection of multiple target sequences in a single cell can present significant problems due to the different efficiencies of each primer pair.
  • the method of the present invention utilizes one or more amplification probes and/or amplification sequences, as described above, and the LD-PCR technique to locate and detect in situ target nucleic acid, which offers certain advantages over the RT-PCR method.
  • the secondary structure of the target RNA does not affect the outcome of the assay.
  • the crosslinking of target nucleic acids and cellular proteins due to tissue fixatives, as discussed above, does not interfere with the amplification of probe sequences since there is no primer extension of the target RNA as in the RT-PCR method.
  • amplification probes according to the present invention may be designed such that there are common primer-binding sequences within probes detecting different genotypic variants of a target pathogen. This enables the assay to detect multiple targets in a single sample.
  • the assay may utilize two or more amplification probes according to this method to detect HCV RNA and ⁇ -actin RNA, whereby the ⁇ -actin probe serves as an internal control for the assay.
  • primer-binding sequences in the probe may be designed to (1) minimize non-specific primer oligomerization and (2) achieve superior primer- binding and increased yield of PCR products, thereby increasing sensitivity of the assay.
  • amplification probe that undergoes PCR circularizes after binding to target nucleic acid to become a circular molecule, multimeric products may be generated during polymerization, so that amplification products are easily detectable.
  • An in situ LD-PCR assay to detect target nucleic acids in histological specimens utilizes a ligation dependent full length amplification probe, and involves the following steps:
  • Sample tissue e.g. liver, that may be frozen, or formalin-fixed and embedded in paraffin, is sectioned and placed on silane-coated slides.
  • the sections may be washed with xylene and ethanol to remove the paraffin.
  • the sections may then be treated with a proteolytic enzyme, such as trypsin, to increase membrane permeability.
  • the sections may be further treated with RNAase-free DNAase to eliminate cellular DNA.
  • An amplification probe may be suspended in a suitable buffer and added to the sample sections on the slide and allowed to hybridize with the target sequences.
  • the probe may dissolved in 2 x SSC with 20% formamide, added to the slide, and the mixture incubated for 2 hours at 37°C for hybridization to occur.
  • the slide may be washed once with 2 x SSC and twice with 1 x ligase buffer before DNA ligase may be applied to the sample.
  • lU/20 ⁇ l of the ligase enzyme may be added to each slide, and the mixture incubated at 37°C for 2 hours to allow circularization of the probe.
  • the slide may be washed with 0.2 x SSC (high stringency buffer) and 1 x PCR buffer to remove unligated probes before the next step of amplification by PCR.
  • the PCR reaction mixture containing the amplification primers and one or more labelled nucleotides is now added to the sample on the slide for the amplification of the target sequences.
  • the label on the nucleotide(s) may be any signal generating moiety, including, inter alia. radioisotopes, e.g.. 32 P or 3 H, fluorescent molecules, e.g.. fluorescein and chromogenic molecules or enzymes, e.g. f peroxidase, as described earlier.
  • Chromogenic agents are preferred for detection analysis, e.g.. by an enzyme linked chromogenic assay.
  • digoxinin-labelled nucleotides are utilized.
  • the PCR product, tagged with digoxinin-labelled nucleotides is detectable when incubated with an antidigoxinin antibody-alkaline phosphatase conjugate.
  • the alkaline phosphatase-based colorimetric detection utilizes nitroblue tetrazolium, which, in the presence of 5- Bromo-4-chloro-3-indolylphosphate, yields a purple-blue precipitate at the amplification site of the probe.
  • the ligation and the PCR amplification step of the in situ LD-PCR detection method can be carried out simultaneously and at a higher temperature, by using a thermostable ligase enzyme to circularize the amplification probe.
  • further embodiments of in situ LD-PCR may utilize amplification probes that are designed to detect various genotypic variants of a pathogen e.g. HCV, that are based on the known HCV sequences of these variants (Stuyver et al., 1993, J.Gen. Vir. 74: 1093-1102).
  • different type-specific probes may be added together to the sample, and detection of HCV sequences and amplification of the probe sequences carried out by in situ LD-PCR as described above.
  • the amplified probe sequences are assayed for the presence of individual variant genotypes by in situ hybridization with type specific internal probes that are labelled to facilitate detection.
  • the target nucleic acid sequence may be directly detected using the various amplification probes and/or amplification sequences described above, without amplification of these sequences.
  • the amplification probes and/or amplification sequences may be labelled so that they are detectable.
  • Reagents for use in practicing the present invention may be provided individually or may be packaged in kit form.
  • kits might be prepared comprising one or more first, e.g.. capture/amplification-1 probes and one or more second, e.g.. amplification-probe-2 probes, preferably also comprising packaged combinations of appropriate generic primers.
  • Kits may also be prepared comprising one or more first, e.g..
  • kits may be prepared comprising one or more first, e.g.. capture/amplification-1 probes and one or more second, full length, ligation-dependent circularizable probes, e.g. f amplification-probe-2.
  • Such kits may preferably also comprise packaged combinations of appropriate generic primers.
  • other reagents required for ligation e.g.. DNA ligase
  • Additional reagents also may be included for use in quantitative detection of the amplified ligated amplification sequence, e.g.. control templates such as an oligodeoxyribonucleotide corresponding to nanovariant RNA.
  • kits may include reagents for the in situ detection of target nucleic acid sequences e.g. in tissue samples.
  • the arrangement of the reagents within containers of the kit will depend on the specific reagents involved. Each reagent can be packaged in an individual container, but various combinations may also be possible.
  • Capture/Amp-probe-1 HAV
  • Amp-probe-2 HAV
  • Capture/Amp-probe-1 HAV
  • Capture/Amp-probe-1 is an oligodeoxyribonucleotide comprising 59 nucleotides and a 3' biotin moiety, which is added by using a 3'- biotinylated nucleoside triphosphate as the last step in the synthesis.
  • the Capture/Amp-probe-1 (HIV) used in this Example has the following nucleotide sequence (also listed below as SEQ ID NO. 1) :
  • nucleotides at positions 24-59 comprise the generic 3' end of the probe. Within this region are recognition sequences for Sjnal (CCCGGG) , EcoRI (GAATTC) and Hindlll (AAGCTT) at nucleotides 41-46, 46-51 and 52-57, respectively.
  • the 5' portion of the sequence comprising nucleotides 1-23 is complementary and hybridizes to a portion of the gag region of HIV-1 RNA.
  • Amp-probe-2 is a 92 nucleotide oligodeoxyribonucleotide which has the following sequence (also listed below as SEQ ID NO. 2) :
  • nucleotides at positions 71-92 comprise the 3' specific portion of this probe, complementary and hybridizable to a portion of the gag region of HIV-1 RNA immediately adjacent to the portion of the gag region complementary to nucleotides 1-23 of Capture/Amp-probe-1 (HIV) .
  • Nucleotides 1-70 comprise the generic 5' portion of Amp-probe-2 (HIV) . Ligation of the 5' end of Capture/Amp-probe-1
  • HIV ligated amplification sequence
  • This ligated amplification sequence is 151 nucleotides long, which provides an ideal sized template for PCR.
  • the generic nucleotide sequences of the ligated amplification sequence (HIV) comprising nucleotides 116-135 (derived from nucleotides 24-43 of Capture/Amp-
  • nucleotides 1-70 correspond in sequence to nucleotides 1-90 of the (-) strand of the WSI nanovariant RNA described by Schaffner et al. , J. Molec. Biol. 117:877-907 (1977).
  • WSI is one of a group
  • Primer-1 which has a length of 21 nucleotides, is complementary to the 3 ' sequence
  • Capture/Amp-probe-1 HAV 35 of Capture/Amp-probe-1 (HIV) (nucleotides 38-58) and has the sequence (also listed below as SEQ ID NO. 5) :
  • Primer-2 which has a length of 20 nucleotides, corresponds in sequence to the 5' sequence of Amp- probe-2 (HIV) (nucleotides 1-20) and has the sequence (also listed below as SEQ ID NO. 6) :
  • Target HIV-1 RNA (100 ⁇ l) is dissolved in an equal volume of lysis buffer comprising 5M GnSCN, lOOmM EDTA, 200mM Tris-HCl (pH 8.0), 0.5% NP-40 (Sigma Chemical Co., St. Louis, MO), and 0.5% BSA in a 1.5 ml microfuge tube.
  • lysis buffer comprising 5M GnSCN, lOOmM EDTA, 200mM Tris-HCl (pH 8.0), 0.5% NP-40 (Sigma Chemical Co., St. Louis, MO), and 0.5% BSA in a 1.5 ml microfuge tube.
  • HIV HIV
  • SEQ ID NO. 1 SEQ ID NO. 1
  • Amp-probe-2 SEQ ID NO. 2
  • streptavidin-coated paramagnetic beads obtained from Promega Corp.
  • HAV HIV /paramagnetic beads
  • a magnetic field generated by a magnet in a microfuge tube holder rack was applied to the complex to retain it on the side of the reaction tube adjacent the magnet to allow unbound material to be siphoned off.
  • the complex was then washed twice with a 1.5M GnSCN buffer to remove any unbound proteins, nucleic acids, and probes that may be trapped with the complex.
  • the magnetic field technique facilitated the wash steps.
  • the GnSCN then was removed by washing the complex with 300mM KCl buffer (300mM KCl, 50mM Tris-HCl, pH 7.5, 0.5% Non- IDEP-40 lmM EDTA) .
  • the two probes were then covalently joined using T 4 DNA ligase (Boehringer Manheim) into a functional ligated amplification sequence (HIV) (SEQ ID NO. 3) , which can serve as a template for PCR amplification.
  • the ligation reaction was carried out in the presence of a IX ligation buffer comprising a 1:10 dilution of 10X T 4 DNA ligase ligation buffer (660mM Tris-HCl, 50mM MgCl 2 , lOmM dithioeryritol, lO M ATP - pH 7.5 at 20°C) obtained from Boehringer Manheim.
  • paramagnetic beads containing bound ligated amplification sequence were washed with IX T 4 DNA ligase ligation buffer and resuspended in 100 ⁇ l of IX T 4 DNA ligase ligation buffer. 20 ⁇ l of bead suspension was removed for the ligation reaction. 2 ⁇ l T DNA ligase was added to the reaction mixture, which was incubated at 37°C for 60 minutes.
  • PCR amplification of the bound ligated amplification sequence 80 ⁇ l of a PCR reaction mixture comprising Taq DNA polymerase, the two generic PCR primers (SEQ ID NOS. 5 and 6) , a mixture of deoxynucleoside triphosphates and 32 P-dCTP was added to the ligation reaction.
  • a two temperature PCR reaction was carried out for 30 cycles in which hybrid formation and primer extension was carried out at 65°C for 60 seconds and denaturation was carried out at 92°C for 30 seconds.
  • nanovariant DNA SEQ ID NO. 4
  • Fig. 3, Lane A As a control, nanovariant DNA (SEQ ID NO. 4) was also subjected to 30 cycles of two temperature PCR, under the same conditions as for the ligated amplification sequence (HIV) , electrophoresed and autoradiographed (Fig. 3, Lane B) .
  • the amplified ligated amplification sequence (HIV) migrated in a single band (151 nucleotides) at a slower rate than the amplified nanovariant DNA (90 nucleotides) .
  • HIV-1 RNA transcript Streptavidin-coated paramagnetic beads, 3'-biotinylated Capture/Amp-probe-1 (HIV) (SEQ ID NO. 1), Amp-probe-2 (HIV) (SEQ ID NO. 2) 5' ( 32 P) , HIV-1 RNA transcript.
  • Streptavidin-coated paramagnetic beads 3 '-biotinylated Capture/Amp-probe-1 (HIV), Amp-probe-2 (HIV) 5' ( 32 P) . 3. Streptavidin-coated paramagnetic beads,
  • HIV-1 RNA transcript A Ammpp--pprroobbee--22 (HIV) 5' ( 32 P) , HIV-1 RNA transcript.
  • GnSCN buffer comprising 1M GnSCN, 0.5% NP-40 (Nonidet P-40, N-lauroylsarcosine, Sigma Chemical Co., St Louis, MO), 80mM EDTA, 400mM Tris HCl (pH 7.5) and 0.5% bovine serum albumin.
  • the reaction mixtures were incubated at 37°C for 60 minutes. After incubation, the reaction mixtures were subjected to a magnetic field as described in Example 1 and washed (200 ⁇ l/wash) two times with 1M GnSCN buffer and three times with a 300mM KCl buffer comprising 300mM KCL, 50mM Tris-HCl (pH 7.5), 0.5% NP- 40 and ImM EDTA.
  • rRNA is an essential constituent of bacterial ribosomes; 2) comparative analysis of rRNA sequences reveals some stretches of highly conserved sequences and other stretches having a considerable amount of variability; 3) rRNA is present in large copy numbers, i.e. 10 3 to 10 4 molecules per cell, thus facilitating the develop ⁇ ment of sensitive detection assays; 4) the nucleotide sequence of 16S rRNA can be rapidly determined without any cloning procedures and the sequence of most Mycobacterial 16S rRNAs are known.
  • Three pairs of Capture/Amp-probe-1 and Amp-probe-2 probes are prepared by automated oligonucleotide synthesis (as above) , based on the 16S rRNA sequences published by Boddinghaus et al. , and Rogall et al.
  • the first pair of probes (MYC) is generic in that the specific portions of the first and second probes are hybridizable to 16S RNA of all Mycobacteria spp; this is used to detect the presence of any mycobacteria in the specimen.
  • the second pair of probes (MAV) is specific for the 16S rRNA of M. avium
  • the third pair of probes (MIN) is specific for the 16S rRNA of M.
  • the probes that may be used for generic detection of all Mycobacter spp. comprise a first and second probe as in Example 1.
  • the first probe is a 3' biotinylated - Capture/Amp-probe-1 (MYC) , an 5 oligodeoxyribonucleotide of 54 nucleotides in length with the following sequence (also listed below as SEQ ID NO. 7) :
  • Nucleotides 1-18, at the 5' end of the probe are complementary to a common portion of Mycobacterial 16S rRNA, i.e. , a 16S rRNA sequence which is present in all
  • the 3' portion of the probe, 20 comprising nucleotides 19-54 is identical in sequence to the 36 nucleotides comprising the generic portion of
  • the second probe is Amp-probe-2 (MYC) , an oligodeoxyribonucleotide of 91 nucleotides in length, 25 with the following sequence (also listed below as SEQ
  • Nucleotides 71-91 at the 3' end of the probe are complementary to a common portion of 16S rRNA adjacent 40 the region complementary to nucleotides 1-18 or
  • Capture/Amp-probe-1 disclosed above, common to all Mycobacteria spp. Nucleotides 1-70 at the 5' end of the probe comprise the same generic sequence set forth for Amp-probe-2 (HIV) in Example 1.
  • End to end ligation of the two probes provides ligated amplification sequence (MYC) , 145 nucleotides in length, for detection of all Mycobacteria spp. , having the following sequence (also listed below as SEQ ID NO. 9) :
  • the pair of probes for specific detection of M. avium are as follows:
  • the first probe is a 3' biotinylated-Capture/Amp- probe-1 (MAV) , an oligodeoxyribonucleotide of 56 nucleotides in length with the following sequence (also listed below as SEQ ID NO. 10) :
  • Nucleotides 1-20 at the 5' - end are complementary to a portion of 16S rRNA specific for M. aviu . Nucleotides
  • 21-56 comprise the same generic sequence, as above.
  • the second probe is Amp-probe-2 (MAV) , an oligodeoxyribonucleotide of 90 nucleotides in length, with the following sequence (also listed below as SEQ
  • Nucleotides 71-90 at the 3' end of the probe comprise the specific nucleotide sequence complementary to a region of 16S rRNA specific for M. avium. adjacent the specific sequence recognized by Capture/Amp-probe-1 (MAV) .
  • Nucleotides 1-70 comprise the same generic sequence as above.
  • End to end ligation of the two probes provides a 146 nucleotide long ligated amplification sequence (MAV) for detection of M. avium having the following sequence (also listed below as SEQ ID NO. 12) :
  • the first probe is a 3' - biotinylated Capture/Amp-probe-1 (MIN) , an oligonucleotide of 56 nucleotides in length with the following sequence (also listed below as SEQ ID NO. 13): 1 11 21 31
  • Nucleotides 1-20 at the 5' end are complementary to a portion of 16S rRNA specific for M. intracellulaire. Nucleotides 21-56 comprise the same generic sequence as above.
  • the second probe is Amp-probe-2 (MIN) , an oligodeoxyribonucleotide or 90 nucleotides in length, with the following sequence (also listed below as SEQ ID NO. 14) :
  • Nucleotides 71-90 at the 3' end of the probe comprise the specific nucleotide sequence complementary to a region of M. intracellulaire 16S rRNA adjacent the specific sequence recognized by Capture/Amp-probe-l
  • End to end ligation of the two probes provides a
  • nucleotide long ligated amplification sequence for detection of M. intracellulaire, having the following sequence (also listed below as SEQ ID NO.
  • Pathol. 4_1:687, 1988 After lysis, about 120 ⁇ l of concentrated material is dissolved in an equal volume of the 5M GnSCN buffer of Example 1. The mixture is boiled for 30 minutes, which because of the nature of mycobacterial cell walls, is required for lysis of Mycobacteria spp. The subsequent procedures (i.e.. capture, ligation, PCR and detection) are the same as those employed in Example 1.
  • a direct detection is made by measuring radioactivity representing 32 P-5'- AMP-probe-2 captured on the magnetic beads. After the unbound radioactively-labeled Amp-probe-2 is removed by extensive washing, the target 16S rRNA molecules that are present in concentrations of more than 10 6 /reaction is detectable. Target 16S rRNA that cannot be detected directly is subjected to PCR amplification of the ligated amplification sequences as per Example 1.
  • the primers for use in amplification are the same two generic primers of Example 1 (SEQ ID NOS. 5 and 6) .
  • EXAMPLE 4 DETECTION OF HCV RNA IN A SAMPLE.
  • Hepatitis C virus (HCV) , an RNA virus, is a causative agent of post transfusion hepatitis.
  • HCV has been found to be distantly related to flavivirus and pestivirus and thus its genome has a 5' and a 3' untranslated region (UTR) and encodes a single large open reading frame (Lee et al. , J. Clin. Microbiol. 3_0:1602-1604, 1992).
  • the present method is useful for detecting the presence of HCV in a sample.
  • Capture/Amp-probe-1 HCV
  • Amp-probe-2 HCV
  • Capture/Amp-probe-1 HCV
  • Capture/Amp-probe-1 which is biotinylated at the 3' end, is a 55 nucleotide long oligodeoxyribonucleotide having the following nucleotide sequence (also listed below as SEQ ID NO. 16) :
  • HCV HCV
  • Amp-probe-2 is a 90 nucleotide long oligodeoxyribonucleotide having the following nucleotide sequence (also listed below as SEQ ID NO.
  • Nucleotides 71-90 comprise the 3' specific portion of the probe, complementary and hybridizable to a portion of the HCV 5' UTR immediately adjacent to the portion of the HCV genome hybridizable to nucleotides 1-19 of Capture/Amp-probe-2 (HCV) .
  • Nucleotides 1-70 comprise the same generic sequence as in Amp-probe-2 (HIV) of
  • Example 1 provides a 145 nucleotide long ligated amplification sequence (HCV) for detection of HCV in a sample, having the sequence (also listed below as SEQ ID NO: 1
  • the ligated amplification sequence (HCV) is amplified using a two temperature PCR reaction as in Example 1.
  • the PCR primers used for amplification are the same two generic primers (SEQ ID NOS. 5 and 6) of Example 1.
  • a pair of amplication probes and two capture/ amplification probes were used to assay for and detect
  • HCV RNA in a sample, thereby increasing the capture efficiency of the assay.
  • Capture/Amp- probe-1 (all oligomers described in this Example are designated “(HCV A) " to distinguish from the probes "(HCV)" of Example 4) having SEQ ID NO. 22 and Capture/Amp-probe-lA (HCV A) having SEQ ID NO. 23 are designed and synthesized such that their 5' termini are biotinylated and the 3' region of the probes comprises sequences complementary to and hybridizable with sequences in the 5'UTR of HCV RNA (Fig. 4) .
  • the generic nucleotide sequence at the 5' region of the probes that are not hybridizable to the target nucleic acid sequence are designed and synthesized to have random sequences and a GC content of, at least, 60%, and such that they exhibit minimal secondary structure e.g. hairpin or foldback structures.
  • Capture/Amp-probe-1 which is biotinylated at the 5' terminus, is a 45 nucleotide DNA oligomer, such that nucleotides 5 to 45 in the 3' region, are complementary to and hybridizable with sequences in the
  • the oligomer has the following nucleotide sequence (also listed below as
  • Capture/Amp-probe-lA which is also biotinylated at the 5' terminus, is also a 45 nucleo ⁇ tide DNA oligomer, such that nucleotides 5 to 45 in the 3' region are complementary to and hybridizable with sequences in the 5'UTR of HCV RNA that are immediately adjacent to the region of the 5'UTR of the HCV RNA hybridizable with Capture/Amp-probe-1 (HCV A) and such that the oligomer has the following nucleotide sequence (also listed below as SEQ ID NO. 23) :
  • Amp-probe-2 (HCV A) and Amp-probe-2A (HCV A) each contain a nucleotide sequence complementary to and hybridizable with the conserved 5'UTR of HCV RNA.
  • Amp-probe-2 (HCV A) is a 51 nucleotide oligomer such that nucleotides 1 to 30 in the 5' region are complementary to and hybridizable with sequences in the 5'UTR of HCV RNA, and that the nucleotides 34 to 51 at the 3' terminus bind to and hybridize with PCR primer-3 and such that the oligomer has the following nucleotide sequence (also listed below as SEQ ID NO. 24) :
  • Amp-probe-2A is a 69 nucleotide oligomer such that nucleotides 40 to 69 in the 3' region are complementary to and hybridizable with sequences in the 5'UTR of HCV RNA genome immediately adjacent to the portion of the HVC RNA genome hybridizable to nucleotides 1-30 of Amp-probe-2 (HCV A) and such that the nucleotides 1 to 18 at the 5' terminus bind to and hybridize with PCR primer-4 and such that nucleotides 19 to 36 at the 5' terminus bind to and hybridize with PCR primer-5, and such that the oligomer has the following nucleotide sequence (also listed below as SEQ ID NO. 25) :
  • HCV A ligation- amplification sequence
  • Primer-3 used for the first series of PCR amplification of the ligated amplification sequence, SEQ ID NO. 26 (HCV A) , and which has a length of 18 nucleotides, is complementary to sequence comprising nucleotides 34 to 51 at the 3' terminus of Amp-probe-2 (HCV A) , and is, therefore, also complementary to the sequence comprising nucleotides 103 to 120 of the ligated amplification sequence, SEQ ID NO. 26 (HCV A) , and has the sequence (also listed below as SEQ ID NO. 27) :
  • Primer-4 used for the first series of PCR amplification of the ligated amplification sequence (HCV A), SEQ ID NO. 26, and which has a length of 18 nucleotides, is complementary to the sequence comprising nucleotides 1-18 at the 5' terminus of the Amp-probe-2A (HCV A) , and is, therefore, also complementary to the sequence comprising nucleotides 1 to 18 of the ligated amplification sequence, SEQ ID NO. 26 (HCV A) , and has the sequence (also listed below as SEQ ID NO. 28) :
  • Primer-5 a DNA oligomer of 18 nucleotides is used for the second series of PCR amplification of the ligated amplification sequence (HCV A), SEQ ID NO. 26, such that the primer is complementary to the sequence comprising nucleotides 19-36 of the Amp-probe-2A (HCV A) , and is, therefore, also hybridizable to the sequence comprising nucleotides 19-36 of the ligated amplification sequence SEQ ID NO. 26 (HCV A) , and has the sequence (also listed below as SEQ ID NO. 29) :
  • the assay utilizing the above probes and primers was used to detect HCV RNA in 24 human serum samples that were stored at -70°C until use.
  • 180 ⁇ l serum sample was added to concentrated lysis buffer (prepared by condensing 250 ⁇ l of the lysis solution containing 5M GnSCN, 0.5% bovine serum albumin, 80mM EDTA, 400mM Tris HCl (pH 7.5), and 0.5% Nonidet P-40 so that the mixture of serum and lysis buffer would have a final concentration of 5M GnSCN) mixed well and incubated for 1 hour at 37°C to release the target RNA from HCV particles.
  • concentrated lysis buffer prepared by condensing 250 ⁇ l of the lysis solution containing 5M GnSCN, 0.5% bovine serum albumin, 80mM EDTA, 400mM Tris HCl (pH 7.5), and 0.5% Nonidet P-40 so that the mixture of serum and lysis buffer would have a final concentration of 5M GnSCN
  • hybridization buffer [0.5% bovine serum albumin, 80mM EDTA, 400 mM Tris-Hcl (pH 7.5), 0.5% Nonidet-P40] with 10 10 molecules each of amplification probes, Amp-probe-2 (HCV A) and Amp-probe-2A (HCV A) oligomers, and 10 11 molecules each of capture/amplification probes, Capture/Amp-probe-1 (HCV A) and Capture/Amp-probe-lA (HCV A) .
  • the addition of the hybridization buffer reduced the concentration of the guanidium isothiocyanate (GnSCN) from 5M to 2M to allow the hybridization to occur.
  • GnSCN guanidium isothiocyanate
  • the mixture was incubated at 37°C for 1 hour to let the various probes hybridize with the target RNA, whereupon 30 ⁇ l of streptavidin-coated paramagnetic beads (Promega) were added to the hybridization mixture before incubation at 37° C for 20 minutes to allow ligand binding.
  • the beads were washed with 150 ⁇ l of 2M GnSCN to eliminate any free probes, proteins, extraneous nucleic acids and potential PCR inhibitors from the hybridization mixture; this was followed by the removal of the GnSCN by washing twice with 150 ⁇ l ligase buffer [66mM Tris-Hcl (pH 7.5) lmM DTT, ImM ATP, 0.5% Nonidet P-40 and ImM MnCl 2 ].
  • the magnetic separation of the bound complex from the supernatant was effected by the magnetic field technique described in Example 1.
  • the amplification probes, Amp-probe-2 (HCV A) and Amp-probe-2A (HCV A) , bound to target RNA were then covalently joined to create the ligated amplification sequence (HCV A) that was utilized as a template for PCR amplification.
  • the hybrid complex was resuspended in 20 ⁇ l ligase buffer with 5 units of T 4 DNA ligase
  • first PCR reaction lO ⁇ l of the ligated mixture, including the beads, was added to 20 ⁇ l of PCR mixture [0.06 ⁇ M each of Primer-3 and
  • Primer-4 1.5 Units Taq DNA Polymerase, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 1.5 mM MgCl 2 , lOmM Tris-HCl (pH 8.3) 50mM KCl] and the mixture incubated at 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute, for 35 cycles.
  • 5 ⁇ l of the product was transferred to a second PCR mixture [same components as the first PCR mixture except that Primer-4 was substituted with Primer-5] for "the second PCR reaction" (a semi-nested PCR approach to increase the sensitivity of the assay) carried out under the same conditions as the first PCR reaction.
  • the assay is quantitative over, at least, a range of 10 2 to 10 5 target molecules.
  • HCV A Capture/Amp-probe-1
  • HCV A Capture/Amp-probe-lA
  • Results showed that 25.7% of labelled HCV RNA was retained on the beads when captured by Capture/Amp-probe-1 (HCV A) alone, 35.8% retained with Capture/Amp-probe-lA (HCV A) alone and 41.5% of the target RNA was retained when both the capture probes were used. Therefore the double-capture method was more efficient than the use of a single capture probe.
  • Example 1 uses a capture/amplification probe and a pair of amplication probes to detect the presence of HIV-1
  • RNA Capture/Amp-probe-1 (HIV) , SEQ ID NO. 1 and a pair of amplification probes Amp-probe-2 (HIV A) (all oligomers described in this Example are designated “(HIV A) " to distinguish from the probes "(HIV) " of Example 1) (SEQ ID NO. 19) and Amp-probe-2A (HIV A), (SEQ ID NO. 20) , are utilized such that the generic nucleotide sequences of the ligated amplification sequence (HIV A) (SEQ ID NO.
  • nucleotides 1-26 derived from nucleotides 1-26 of Amp-probe-2 (HIV A) and nucleotides 86-112 derived from nucleotides 40- 65 of Amp-probe-2A (HIV A) are designed and synthesized to have random sequences and a GC content of, at least, 60%, and such that they exhibit minimal secondary structure e.g. hairpin or foldback structures.
  • Amplification probe Amp-probe-2 (HIV A) is a 47 nucleotide DNA oligomer such that nucleotides 27 to 47 in the 3' region, are complementary to and hybridizable with sequences in the gag region of the target HIV-1 RNA, and that the oligomer has the following nucleotide sequence (also listed below as SEQ ID NO. 19) :
  • Amplification probe Amp-probe-2A (HIV A) is a 65 nucleotide DNA oligomer such that nucleotides 1 to 39 in the 5' region, are complementary to and hybridizable with sequences in the gag region of the target HIV-1 RNA, immediately adjacent to the portion of the HIV-1 RNA genome hybridizable to nucleotides 27-47 of the Amp-probe-2 (HIV A) and that the oligomer has the following nucleotide sequence (also listed below as SEQ ID NO. 20) :
  • End to end ligation of the two amplification probes provides a 112 nucleotide ligated amplification sequence (HIV A) such that the sequence serves as a detectable sequence for HIV-1 RNA as well as a template for amplification reactions, and has the following sequence (also known as SEQ ID NO. 21)
  • the assay employs a single ligation independent amplification probe and two capture/amplification probes to detect HCV RNA in a sample.
  • Capture/Amp- probe-1 HCV A
  • Capture/Amp-probe-lA HCV A
  • SEQ ID NO. 30 SEQ ID NO. 30
  • Amp-probe-2 also referred to as amplification sequence, has the following sequence (also listed below as SEQ ID NO. 30) :
  • the capture, detection and the optional amplifica ⁇ tion of the probe sequences was carried out as described in Example 5, except that primers -3 and -4, only, were utilized in a single PCR amplification step, the second PCR step was omitted, and that the ligation step was omitted.
  • the method in this Example employs the two capture/amplification probes Capture/Amp-probe-1 (HCV A) and Capture/Amp-probe-lA (HCV A) described in
  • Example 5 and a single amplification probe, Amp-probe-2 (HCV C) (all oligomers described in this Example are designated “(HCV C) " to distinguish from the probes "(HCV)" of Example 4) that hybridizes to the target nucleic acid and circularizes upon ligation of its free termini as shown in Fig. 7.
  • HCV C Amp-probe-2
  • Amp-probe-2 is a 108 nucleotide amplification probe, also referred to as an amplification sequence, such that nucleotides 1-26 in the 5' terminus of the oligomer are complementary to and hybridizable to a sequence in the 5'UTR of the target HCV RNA (indicated by (a) in Fig. 7) and such that nucleotides 83-108 at the 3' terminus of the oligomer are complementary to and hybridizable to a sequence in the 5'UTR of the target HCV RNA (indicated by (b) in Fig. 7) .
  • HCV C The sequence of Amp-probe-2 (HCV C) is given as follows (also listed as SEQ ID NO. 31) : 5' - CCTTTCGCGA CCCAACACTA CTCGGCTGTC TGTGTATCTG CTAACCAAGA GCAACTACAC GAATTCTCGA TTAGGTTACT GCGCACCCTA TCAGGCAGTA CCACAAGG -3'
  • Primer-3 (SEQ ID NO. 27) , used for the first series of PCR amplification of the ligated and circu ⁇ larized Amp-probe-2 (HCV C) , is an 18 nucleotide long oligomer that is complementary to the sequence comprising nucleotides 27 to 45 of Amp-probe-2 (HCV C) .
  • Primer-4 (SEQ ID NO. 28) , also used for the first series of PCR amplification of the ligated and circularized Amp-probe-2, is a 18 nucleotide long oligomer that is complementary to the sequence comprising nucleotides 46 to 63 of Amp-probe-2 (HCV C) .
  • the assay is significantly quantitative at least over a range of 10 4 to 10 7 target molecules.
  • Sample tissue e.g. liver, that is frozen, or formalin-fixed and embedded in paraffin, is sectioned
  • the sections can be washed with xylene and ethanol to remove the paraffin.
  • the sections are then treated with a proteolytic enzyme such as trypsin (2mg/ml) at 37°C for 4-12 minutes to increase membrane permeability.
  • the sections may be further treated with RNAase-free DNAase to eliminate cellular DNA.
  • Amp-probe-2 HCV C
  • SEQ ID NO. 31 described in
  • Example 8 is dissolved in 2 x SSC with 20% formamide, (10 10 molecules/lOO ⁇ l) , added to the slide and the mixture incubated for 2 hours at 37°C for hybridization to occur.
  • the slide is washed once with 2 x SSC and twice with 1 x ligase buffer before the ligation step.
  • lU/20 ⁇ l of DNA ligase is added to each slide, and the mixture incubated at 37°C for 2 hours to allow circularization of the hybridized probe.
  • the slide is washed with 0.2 x SSC (high stringency buffer) and 1 x PCR mixture (the same as described in Example 5) to remove unligated probe before the sample is subjected to amplification by PCR.
  • the PCR product labelled with the digoxinin, is detectable when incubated with antidigoxinin antibody- alkaline phosphatase conjugate (Boehringer Mannheim) .
  • the alkaline phosphatase-based colorimetric detection method uses the the chromagen nitroblue tetrazolium, which in the presence of 5-Bromo-4-chloro-3- indolylphosphate, yields a purple-blue precipitate at the amplification site of the probe.
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Abstract

L'invention se rapporte à un procédé amélioré de détection rapide, sensible et standardisée, d'un acide nucléique cible à partir d'un micro-organisme pathogène, ou virus, ou gène normal ou anormal. Le procédé consiste à hybrider un acide nucléique cible à plusieurs sondes oligonucléotidiques non superposées qui s'hybrident à des régions adjacentes dans l'acide nucléique cible, ces sondes étant respectivement des sondes de capture/amplification et des sondes d'amplification en présence de perles paramagnétiques recouvertes d'une fraction de liaison de ligand. Par la liaison d'un ligand fixé à une extrémité de la sonde de capture/amplification et par l'hybridation spécifique de parties des sondes à des séquences adjacentes de l'acide nucléique cible, on obtient un complexe comprenant l'acide nucléique cible, les sondes et les perles paramagnétiques. Les sondes peuvent être ensuite soudées pour former une séquence d'amplification, soudée, contiguë, liée aux perles, qui, lors de la dénaturation visant à retirer l'acide nucléique cible et les sondes non soudées, peuvent être directement détectées ou amplifiées par une technique appropriée d'amplification, telle que l'ACP, utilisée dans la détection. La détection de la séquence d'amplification soudée, s'effectuant soit directement, soit après son amplification, indique la présence de l'acide nucléique cible dans un échantillon.
EP95923925A 1994-06-22 1995-06-14 Amplification dependant de la liaison s'appliquant a la detection d'agents pathogenes infectieux et de genes anormaux Withdrawn EP0717782A1 (fr)

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