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
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
• • 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
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
• • 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

Definitions

• This invention relates to disease diagnosis and particularly to a non-radioactive method and apparatus in the form of diagnostic materials which are part of a diagnostic kit and a product which is used to diagnose the direct presence in a sample of a living disease organism, such as the AIDS virus, HSV, CMV, HBV , HAV and FLU viruses, and bacteria such as cholera, mycoplasmas, chlamydia and other invasive body organisms which cause disease, and which are usually infectious.
• a living disease organism such as the AIDS virus, HSV, CMV, HBV , HAV and FLU viruses
• bacteria such as cholera, mycoplasmas, chlamydia and other invasive body organisms which cause disease, and which are usually infectious.
• Viral diagnostic methods are utilized by doctors and clinical laboratories and hospitals and diagnostie aids have generally been developed by these organizations personnel. DNA 'related techniques are not being widely practiced outside research laboratories. Molecular techniques for viral diagnosis are urgently needed, providing they are sensitive, specific, rapid and easy to use. The development of such techniques and diagnostic kits will make it easier to be employed at the doctor's office or at clinical laboratories.
• R1-R9 The clinician uses methods for viral diagnosis are laborious, time consuming or their sensitivity is in question (R1-R9).
• R1 and R2 refer to the references identified in the Appendix hereto, as the designation (R%) identifies all references there identified. These references are specifically incorporated in there entirety herein.
• radioactive probes R22
• This prior attempt to detect a virus by DNA hybridization differs from the present inventions in a number of important respects, even if it were modified in light of the description of other aspects of my inventions.
• the use of radioactive probes is not suitable for use in doctor's offices or clinical laboratories and hospitals and offers risks which are not tolerable, as the risk of personal safety makes such detection unsuitable.
• the preferred embodiment of my inventions provide a safe non-radioactive identifier product probe which will directly link (hybridize) to a virus or bacterial or other organism's DNA or rRNA. This linkage is used for indicating the presence or absence of the suspected invasive organism.
• the probe or the identifier product is a DNA fragment or oligodeoxyribonucleotide complementary to the viral genome or to the rRNA sequence in other organisms, such as bacteria. This fragment would be supplied to a reporter or marker molecule and a marked probe created as the non-radioactive identifier product probe. In my preference this molecule would be fluorophof e. Thus the signal of the reporter or marker identifer product probe would be visualized by ultraviolet light.
• This probe-fluorophope linked product is used to identify chlormetrically the presence thereof on a matrix where the genome or the rRNA of the organism obtained from a clinical sample is immobolized on a matrix.
• the methods elaborated in the detailed description are also be subject to automation for sample manipulation and in the recording of the nonradioactive chlormetrlc/fluorecent signal.
• Representative of what DNA linked products may identify are the AIDS virus, the HSV, CMV, HBV , HAV and FLU viruses and other bacterial organisms such as mycoplasmas, chlamydias, cholera and other invasive organisms.
• the method of diagnosis includes providing in the form of a diagnostic kit and method of its use as way that the suspect organisms can be linked to a specific probe, such as a virus or bacterial protein and thus identified and used for having the identification specifically and non-radioactively visualized by light emission and color identification.
• a specific probe such as a virus or bacterial protein
• Figure 2(a) is a schematic representation of a probe's creation, while Figure 2(b) shows schematically how a sandwich matrix is created, and,
• Figure 3 shows a sample target organism
• Figure 4 shows a marked target organism
• Figure 5A represents a slot matrix used in the immobilization method
• Figure 5B represents a sandwich fragment matrix used in the sandwich method
• Figure 6A represents schematically a method of obtaining the target marked hybrid by the sandwich method
• Figure 6B represents an alternative way of obtaining the marked target material by the sandwich method
• Figure 7 is a schematic sectional view of a probe dispensing device.
• the main objective of the inventions described herein is to provide viral diagnostic kits to be used at the doctor's office or in a clinical laboratory or a hospital.
• Representative of the group of kits which may be made by those skilled in the art after a full understanding of the present inventions are those described herein including developing diagnositc kits for 1) AIDS, 2) HSV, 3) CMV, 4) HBV and HAV and 5)FLU viruses as well as bacteria and other organisms, including those described herein.
• the application of the inventions has use in the field of diagnosis and has potential not only on viral and bacterial diseases diagnosis in man but also in animals, plants and in testing industrial microbial contaminations.
• the target molecule would be ribosomal RNA (rRNA) since these cells contain ribosoraes and thus the target rRNA molecules would be amplified thousands of times.
• rRNA ribosomal RNA
• DNA/RNA genome detection
• the diagnostic kits have b een pro vide d to meet the fol lowing main criteria: 1) nonradioactive, 2) sensitivity, 3) specificity, 4) speed, 5) easy to use, and 6) cost effective.
• HTLV-III is the etiological agent for acquired immune deficiency syndrome (AIDS).
• AIDS acquired immune deficiency syndrome
• the test does not reveal whether an antibody-positive individual has AIDS or even if a person harbor the virus itself and is capable of transmitting the disease to others. Moreover, it is understood that this test is not 100 percent accurate, and that FDA Commissioner Frank Young has recently estimated that about 17 percent of the individuals who test positive for the virus will be "false positives". Some health specialists also are understood to have stated that not everyone who is infected with the virus will have antibodies, and accordingly the test would fail to identify these individuals. Presently, antibody tests for AIDS, are believed to give confusing results because of inadequate fractions, or infections which have not created antigens for one reason or another.
• the preferred type of tests may be considered “second generation tests" since they detect the virus genetic machinery directly. Therefore, this type of testing may be employed as an essential confirmatory test or it may be employed as the first diagnostic test.
• kits and procedures described herein are for use in the doctor's office, as well as in clinical laboratories and hospitals, and before and after reaching the blood bank.
• Herpes Simplex Virus results in Herpes genitalis which has become an increasingly prevalent sexually transmitted disease (CCD report, 1982). It is estimated that 1 in 20 adults in USA were infected annually. Rapid and accurate diagnosis of HSV infection is desirable to permit counseling, management of pregnancy, and the use of newly available chemotherapeutic agents.
• HSV culture is the standard diagnosis test, but is expensive, time consuming, and unavailable in many diagnosis laboratories. Cytological or histopathological diagnosis is helpful in some cases, but the sensitivity of such examinations is generally 50 percent or less (R21).
• the serologic tests now readily available do not even distinguish prior nongenital HSV infection from genital herpes (R22).
• R22 radioactive probes
• This prior attempt to detect as virus by DNA hybridization differs from the present inventions in a number of important respects.
• the use of radioactive probes is not suitable for use in doctor's offices or clinical laboratories and hospitals and offers risks which are not tolerable.
• Viral Hepatitis is a major public health problem occurring endemically in all parts of the world. In man hepatitis A, hepatitis B and yellow fever virus are the most important causes of acute inflammation and necrosis of the liver. Inflammation of the liver is also associated with other viruses such as cytomegalovirus (CMV) and Epstein-Barr (EB). However, the term viral hepatitis commonly referred to hepatitis caused by virus type A and B (R23).
• CMV cytomegalovirus
• EB Epstein-Barr
• HBV Hepatitis B Virus
• R19 Hepatitis B Virus
• R22 human hepatocellular carcinoma
• R7 nuclear and cytoplasmic fractions of infected human cells
• HAV Hepatitis A Virus
• R11.R23 The complete RNA genome of the human HAV has been published including its gene organization (R27).
• CMV Cytomegalovirus
• CMV infection presently depends on detection of a serological rise in titer of antibody to
• CMV antigen or isolation of virus in tissue culture Although urine cultures of some congenitally infected infants may be identified as positive within several days, many cultures for CMV take six weeks or longer before being identified as positive.
• CMV DNA was detected in urine specimen using 32p-labelled DNA probes (R8, R28, R32). This probe detection method differs from the present inventions.
• FLU strains may be indentified by their genomes by use of the method according to my inventions. This will be useful in screening for specific strains.
• the approach used in the direct diagnosis of a suspect sample and in maximizing the resultant signal indicating detection result in a new non-radioact ⁇ ve direct diagnosis method includes the described embodiments of the inventions which involve:
• V The creation ⁇ f a visualization effect for identification of the organism-probe identified product.
• the preferred embodiments result in simplifying the procedures for the manipulation of the samples.
• a DNA probe is basically labeled fragment of DNA and is complementary to the target (e.g. virus) genome.
• target e.g. virus
• Biotin-labelled DNA has been utilized for visualization of bacterial DNA.
• This method is based on the fact that biotin (vitamin H) can strongly bind (non covalent) with egg white glycoprotein (MW68,000) or the non gycosylated protein strepavidin (MW60,000) produced by streptomyces avidini which will lead to the formation of a high affinity complex (Kd is about 10 -15 ) (R45).
• This complex can easily be visualized/by enzymes that are commercially available (e.g. peroxidase, and alkaline phosphatase).
• This method is based on: a) the fusion of an enzyme (e.g. peroxidase, alkaline phosphatase) to a synthetic polyamine carrying many primary amino groups (poythyleneamine) using benzoquinone as crosslinking reagent, b) the modified enzyme can now be able to bind electrostatically to polyaneon (DNA) due to its small, positively charged tail, and c) the ionic binding between the macromolecular components is essential for a successful crosslinking reaction leading to covalent bonds.
• the enzyme could be covalently bound to DNA using gluteraldehide.
• the enzyme(s) is visualized then by standard enzyme reactions.
• Fluorescent Conjugated DNA is employed in a preferred embodiment of my invention. This method is very promising as optimized and modified for non-radioactive diagnostic purposes. This embodiment utilizes fluorescent tagged DNA probes for diagnostic application. This method was employed by others in an experiment to automate DNA sequence analysis (R55). It is based on the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' end. In accordance with the best mode of my invention, this amino group is utilized to synthesize several fluorescent derivatives in accordance with my invention. I have modified this method to make it applicable for non-radioactive diagnosis of viral DNA and for creation of specific probes. This approach is used to produce multiple viral diagnosis kits by attaching different fluorescent groups producing different colors for different probes thereby detecting different pathogens in to the same sample.
• the dot blot method simply immobilizes the samples nucleic acids by blotting directly on the membranes or by filtering the sample through the membrane while the samples DNA are retained by the membrane filter (R59, R60).
• This method has some advantages in as far as it can employ a large number of samples simultaneously. It does not require elaborate purification of samples' nucleic acids and it does not require prior electrophoresis. While this method has been used to retain DNA samples in research it is not known to have been used as an element for a method and apparatus for diagnosis of disease bodies in a sample. It may be employed as an alternate embodiment for. the broad aspects of my inventions.
• Viral genome has been detected in clinical samples utilizing this method using radioactive labelled probes (R8).
• This sandwich method was modified recently by using resin (sephicryl S-500) to immobilize the DNA instead of membrane support (R63).
• the sandwich hybridization method has to be modified to use nonradioactive probes and to be more suitable for crude clinical samples. It is also changed to increase the efficiency of binding DNA molecules by providing solid support, preferably in the form of membranes.
• solid support preferably in the form of membranes.
• membranes There exist suitable commercial membranes for binding DNA and RNA. Nylon membranes could be modified to increase their binding abilities of macromolecules (R64). Such membranes may be used to give colormatic reading as a step for automation of the diagnosis process.
• Resins s uch as s ephi cro yl S-500 may be use drete rna tiv ely .
• Viral genomes has been detected by DNA hybridization in clinical samples such as urine (R8,R28,R32), stool (R10, R65 , R66), cotton swab specimens (R22), mouthwashes (R67), blood serum (R11,R68), blood leucocytes (R69), and semen (R70). It has become evident to me from the recent published work that virus genomes can be detected in clinical samples. However, further improvements of the sample manipulation are required. The followings are examples of alternative sample manipulation methods to be used.
• the method of diagnosis employs a diagnosis kit which employs specifically developed probes for the virus or other disease body and employs a modified sandwich method or alternatively a dot blot method in such a way that the DNA fragment(s) or rRNA complimentary to the suspect genome is chemically linked to a solid support matrix.
• the sandwich method is more suitable for crude clinical samples and is the most preferred mode at the present time.
• the sandwich method is modified such that DNA fragment (s) (oligodeoxyribonucleotides) complementary to the virus genome is chemically linked or crosslinked to a solid support.
• DNA fragment (s) oligodeoxyribonucleotides
• the DNA may be crosslinked to membranes by ultraviolet light. This increases efficiency and sensitivity of detection providing the support membrane or resin is treated afterwards to minimize any non-specific background noise.
• the dot slot hybridization method may be used to detect viral genomes in crude extract.
• the direct dot blot method may be used in applying sample nucleic acids to filter membranes, and this method may be used to allow multiple samples to be tested simultaneously.
• the sandwich method is modified for this purpose. Multiple viral genomes in the same sample may be detected by the sandwich method by employing different immobilized DNA fragments on the same membrane in different slots and by the use of different probe fragments in the hybridization mix. It is frequent for imunosuppressed patients and other patients (such as AIDS victims) to have multiple infections, and accordingly, the use of different hybridization fragments in the same mixture allows multiple screening.
• a probe fragment (rRNA or DNA) complementary to the desired genome prepared and is non-radioac tively labeled and packaged seperately as a part of the diagnostic kit.
• diagnosis is obtained by mixing the probe fragment with the sample genome and the solid support (with attached c omp lementary fragment). If there is a chemical bond established in a hybridization reaction, the signal will develop, but, because a chemical bond is required, this will develop only if the sample has a viral or other body's genome compl eme ntar y to both f ragments .
• the signal will develop only if the sample has a viral genome complementary to both DNA fragments.
• hybridization is carried out by using the probe and the immobolized genome direction in a hybridization reaction.
• the signal will develop if the viral genome complementary to the probe fragment is present.
• the non-radioactive probe in the preferred embodiment of my invention is created by preparing a desired complementary probe fragment or oligonucleotide with an automatic DNA synthesizer and covalently attaching fluorophores to the chemically synthesized oligonucleotide(s) that is complementary to the target genome.
• Automatic DNA synthesizers which can created a suitible probe fragment (s) such as a probe chain AGCTACCGAA (or like chain characteristic of a chain or the target species or strain) can be used in the creation of the probe chain.
• These devices are commercially available from suppliers such as Beckman Instruments, Inc., P.O.Box 10200, Palo Alto, California 94304, U.S.A.
• Suitable fluorophores are commercially available from Aldrich Chemical Co. 940 West Saint Paul Ave. Milwaukee, Wisconsin 53233 U.S. A and Sigma Chemical Company, P.O. Box 14508, St. Louis, Mo. 63170, U.S.A.
• the base composition of DNA Deoxyribonucleic aci d
• rRNA Ribo nucleic aci d
• the genome of organisms each have the specific base composition of a sequence of deoxyribonucleotides or ribonucleotides linked with phospho diester bonds usually in double strands. The strands are stabilized together by hydrogen bonds.
• the matrix is exposed to the sample and the to be indentified sequence denatured and left on the matrix.
• the matrix which is preferred membrane filters that retain DNA/RNA and resist damage such as nylon membranes or . modified nylon membranes to increase their efficiency in binding nuclear acids. These filters are commercially available from Schleicher & Schuell, Keen, New Hampshire 03431 U.S.A., or Bio-Rad, Chemical Division, 2200 Wright Avenue, Richmond, California 94804 or from other companies such as NEN or Amersham for example.
• the resin sephicryl S-500 and like resins are preferred for automation. Sephicryl resins may be obtained from Pharmacia, Inc., 800 Centennial Avenue, Piscataway, New Jersey 08854 U.S.A.
• This matrix is packaged as a part of the kit when the sandwich method is utilized DNA fragment (oligonibonucleotides), is crosslinked to the membrane (R73), the membrance is then treated to minimize back ground noise.
• the probe is placed in a aqueous solution, and in a capsule which can be penetrated by a plunger dispenser.
• a plunger dispenser An example of such a dispenser is shown in Figure 3 in which the dispenser is in the form of a hypodermic syringe which has a capsule penetrating point 31 on the needle's syringe end.
• the capsule under pressure from the syringe plunger forces the capsule against the penetrating point to rupture the capsule which is dispensed through the needle onto the matrix in a hybridization medium.
• This hybridization step is done in sealed plastic hags.
• the sandwich method it is also intended to supply the matrix prepared and presented as above in the sealed bag that contains the hybridization medium and the probe(s).
• the sample then is injected in the bag which is then resealed to accomplish hybridization.
• the matrix is then exposed to UV light, and if fluorescence occurs the target is identified by that fluorescence of the hybrid identified product resulting from the target-probe bond.
• Figure 1 represents the overall method steps.
• block 11 represents the steps involved in creation of a fluorescent-bonded probe
• step 12 represents the step of packaging the fluorescent-bonded probe.
• Step 13 involves obtaining the sample, for example a source of blood 14 or urine 15.
• Step 16 represents the step of obtaining the sample target organism.
• Step 17 represents the step of applying the target organism to the matrix container and fixing the target organism to the matrix.
• Step 18 is the step of mixing the marked-bonded probe such as the preferred fluorophore or halogen marked probe (or other form of probe, having color staining or visualization creation) with sample on the matrix and if a bonding reaction occurs creating a mutation hybrid probe target bond.
• Step 19 is the step of the method under which visualization by the eye (10) occurs, as in the instance illustrated by a simple microscope (20) and ultraviolet light source (21).
• Figure 2 represents a schematic representation of the creation of a probe such as a DNA fragment, oligonucleotide, or oligodeoxyribonucleotide represented as probe base AGATACCGAA (22), and the creation of a fragment thereof ATACCG (23), after elimination of the underscored AG and AA .
• This fragment is mixed with color identification or light emitting reporter or marker molecule source material, such as the preferred flurophore (24), for example F-lurescein isothiocyanate, NBD-F and a marked probe fragment (F)ATACCG
• FIG. 29) for the sandwich method creates a bonding base matrix (30) for the target organism.
• Figure 3 shows a sample target organism being obtained, for example from a blood sample or urine sample (31) by a nucleic acid concentration process (32) to create nucleic acid sample material (33).
• the target organism is for example GCTACCGATGTCGGTAT (33') and in the instance illustrated is found in the sample material (33).
• the step 17 representing the application of the target organism to the matrix and fixing the target organism to the matrix differs depending upon the method employed.
• the sandwich method is illustrated at the left of the dotted vertical line while the immobolization method is illustrated at the right of the dotted vertical, line.
• the matrix (29) has a bonding fragment (30') attached to the matrix to form the bonding matrix (30), and in the dot method example of the immobilization method there is direct bloting attachment of the sample GCTACCGATGTCGGTAT (33') to the matrix (29).
• hybridization material and the reporter gr marker probe are also provided as illustrated schematically by the vertical dotted line and marked probe (25) introduction step (34).
• the probe (25) is introduced into container having the sample matrix bound organism (33') which in this instance is the target chain, a hybridization bonding of the fragment (25) and the organism (33') occurs, creating the target organism which is marked for visualization effect by the flurophobe labeled probe-target bond (36).
• Figure 4A and 4B show in detail the attachment of the marked probe to the sample target organism and to the matrix, as represented at the bottom of Figure 3. This is obtained by supplying the target bearing matrix with the flurophore marked probe GCCATTA(F). A hybridization medium is utilized to provide the environment in which hybridization of the marked probe and the target genome occurs. If there is a match the hybrid reaction occurs and the bonding (36) labels the target (33') on the matrix. The sample target organism is washed and if there is a hybrid reaction which has occrued the marked target will remain on the matrix.
• FIG. 5A and Figure 5B illustrate matrix packages.
• the matrix (29) is packaged in sheets to fit in a slot sample applicator (40).
• a slot sample applicator 40
• FIG. 5A shows the matrix membrane (nylon or resin) (29) between an upper slot bearing surface plate (41) and a backing substrate (42), both made of either plastic or glass.
• FIG. 5B the sandwich method is illustrated.
• a matrix fragment CGTA exists in a coating which is attached to the matrix (29).
• the matrix is then preferably pretreated to minimize any background noise and dried and packaged for individual sample manipulation.
• it can be packaged in plastic bags shown by dotted line (45) containing the hybridization media mix or .in plastic bags wich are seperate from the plastic bag which carries the hybridization mix.
• Figure 6A and Figure 6B represent alternative methods of use of the sandwich method.
• the matrix (29) with bonding fragment coating, if used are packaged in a plastic bag (45) along with, alternatively, the reporter probes (25).
• the plastic bag can also contain the hybridization medium, as well as the probes.
• the sample (33') is injected into the plastic bag, as by a syringe (47), and the bag is resealed.
• the probes in the plastic bag can be introduced at a time after the hybridization medium is added to the bag.
• a matrix with a dried fragment can be packaged in a container (48).
• Hybridization media (49) is provided in a container, as is the probe material (25) all as part of one of the kit packages (50).
• the probes can be mixed, together with the hybridization medium.
• the hybridization medium is prepared, then the prepared sample, and the probes and the hybridization medium, is added to a bag containing the matrix.
• Figure 6A and 6B both permit use of a sealed frangible probe containing container shown in a schematic sectional view of the frangible container (51) for the color producing probe (25) which is inserted in and as shown contained in the dispensing device (52) in Figure 7.
• the probe is sealed in container (51) helps to prevent contamination.
• the dispensing needle (53) has at its base (54) within the syringe dispenser (30) detent points (55) which when forced against (as by the action of the plunger (56)) the container (51) breaks it and allow the syringe to dispense the probe.
• the probe can be contained in another kind of container such as a micropipette with a dispensing stopper plunger, or in a rubber stopped container.
• the matrix may be formed on a microscope slide 22 with cover 23 which can removed to introduce the tagged probe.
• a matrix 24 is positioned within the slide onto which the target material is affixed by introduction and a denaturization process. It will be appreciated by those skilled in the art that various modifications and rearrangements can be made to the apparatus and methods described.
• the probes can be tailored to specific screening criteria. After an understanding of this discription by those skilled in the art such persons will adapt and modify and improve upon the various specific illustrations described herein without departing from the scope of the claimed inventions.
• HBV hepatitis B virus
• Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent. Photobiotin. Nucl. Acids Res. 13, 745- 761.

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  • 1986-12-02 EP EP19870900454 patent/EP0249628A4/de not_active Withdrawn
  • 1986-12-02 JP JP62500190A patent/JPS63502477A/ja active Pending
  • 1986-12-02 WO PCT/US1986/002594 patent/WO1987003621A1/en not_active Application Discontinuation

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