Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-12-12
2001-05-01
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S007100, C435S235100, C536S024300, C536S024310, C536S024320
Reexamination Certificate
active
06225053
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the field of detection assays of viruses in general and more particularly relates to improved detection of hepatitis B virus.
Hepatitis B virus (HBV), formerly termed serum hepatitis, is a member of a group of small DNA-containing viruses that cause persistent noncytopathic infections of the liver, is an infectious agent of humans that is found worldwide and which is perpetuated among humans in a large reservoir of chronic carriers. It is estimated that about 6 to 7% of the earth's population is infected (300 million carriers). HBV infection in humans can cause severe jaundice, liver degeneration and death. HBV enters predominantly by the parenteral route, has a characteristic incubation period of 60 to 160 days, and may persist in the blood for years in chronic carriers. The prevalence of the infection is not uniform throughout the world. There is a geographic gradient in distribution of HBV. It is lowest in North America and Western Europe, where the virus can be detected in 0.1 to 0.5% of the population, and highest in Southeast Asia and sub-Saharan Africa, where the frequency of infection may vary from 5 to 20% of the population. This skewed distribution parallels that of hepatocellular carcinoma and provides strong epidemiologic evidence for an association between chronic HBV infection and this type of malignancy.
Hepatitis B is of great medical importance because it is probably the most common cause of chronic liver disease, including hepatocellular carcinoma in humans. Infected hepatocytes continually secrete viral particles that accumulate to high levels in the blood. These particles are of two types: (i) noninfectious particles consisting of 22 nm spheres and filaments of excess viral coat protein (HBsAg) and containing no nucleic acid (in concentrations of up to 10
13
particles/ml blood) which are referred to as the Australian antigen (AU), and (ii) infectious, DNA-containing particles (Dane particle nucleocapsids) consisting of a 28 nm nucleocapsid core (HBcAg) around which is assembled an envelope containing the major viral coat protein, carbohydrate, and lipid, present in lower concentrations (10
9
particles/ml blood). The human hepatitis B virus is a member of the Hepadna Viridae family, with close relatives including woodchuck hepatitis virus (WHV), duck hepatitis virus (DHV), and ground squirrel hepatitis virus (GHV) (Robinson, 1990). Like retroviruses, the hepadnavirus utilizes reverse transcription of its 3.2 Kb DNA genome (Pugh, 1990). The genome of hepatitis B virus is circular and partially single-stranded, containing an incomplete plus strand. The incomplete plus strand is complexed with a DNA polymerase in the virion which has been shown to elongate the plus strand using the complete minus strand as the template. These morphological and structural features distinguish hepatitis B viruses from all known classes of DNA-containing viruses.
The replication cycle of hepatitis B viruses is also strikingly different from other DNA-containing viruses and suggests a close relationship with the RNA-containing retroviruses. The principal unusual feature is the use of an RNA copy of the genome as an intermediate in the replication of the DNA genome. Infecting DNA genomes are converted to a double-stranded form which serves as a template for transcription of RNA. Multiple RNA transcripts are synthesized from each infecting genome, which either have messenger function or DNA replicative function. The latter, termed “pre-genomes,” are precursors of the progeny DNA genomes because they are assembled into nucleocapsid cores and reverse-transcribed into DNA before coating and export from the cell. Thus each mature virion contains a DNA copy of the RNA pre-genome and a DNA polymerase.
The first DNA to be synthesized is of minus strand polarity and is initiated at a unique site on the viral genetic map. Very small nascent DNA minus strands (less than 30 nucleotides) are covalently linked to a protein, and are likely to act as primer for minus strand DNA synthesis. Growth of the minus strand DNA is accompanied by a coordinate degradation of the pre-genome so that the product is a full-length single-stranded DNA, rather than an RNA:DNA hybrid. Plus strand DNA synthesis has been observed only after completion of the minus strand, and initiates at a unique site close to the 5′ end of the minus strand. Complete elongation of the plus strand is not a requirement for coating and export of the nucleocapsid cores, thus most extracellular virions contain incomplete plus strands and a large single-stranded gap in their genomes. Because the hepatitis virus genome is autonomous and does not utilize a DNA-to-DNA pathway for its replication, continuous intracellular replication of its genome is essential for the maintenance of the virus.
HBV is detected by immunologic techniques such as immune electron microscopy, complement-fixation, immune adherence, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). All blood for transfusion must be screened for HBV to prevent transmission of the virus to blood recipients. Early detection of HBV in infected patients is also important because exposure to blood or objects potentially contaminated with blood or even body excretions may cause infection.
Conventional HBV DNA assays test for the presence of hepatitis B virus genomic DNA in human serum using a full genomic (3.2 Kb) RNA probe. For HBV DNA testing a quantitative assay would be particularly advantageous because the level of HBV DNA in serum correlates with severity of liver disease. A quantitative HBV DNA test would be useful for monitoring chronic carriers of HBV. For example, the effectiveness of antiviral therapy such as that described by Hoofnagle et al.,
J. Hepatol
. 11S:100 (1990), can be assessed more easily with an improved detection assay.
In summary, there is a need for a hybridization assay for clinical diagnosis and quantitative analysis of viral infections, especially HBV, and genetic mutational defects, that is economically feasible for screening large numbers of samples with great sensitivity.
It is therefore an object of the present invention to provide a cost-effective, sensitive, assay for the detection of viral nucleic acids in a sample.
It is a further object of the present invention to provide a cost-effective, sensitive, assay for the quantitation of nucleic acids in a sample.
It is a further object of the present invention to provide an assay in which sample preparation is simple and rapid.
It is a further object of the present invention to provide an assay in which sample preparation does not involve extractions, or other time-consuming purification methods.
It is a further object of the present invention to provide an assay having minimal false positives.
It is a further object of the present invention to provide an accurately quantitative test for monitoring the level of virus in a viral infection.
It is a further object of the present invention to provide a sample for an assay that allows for an assay with a lower detection limit.
It is a further object of the present invention to provide a kit that can be used to screen large numbers of samples for microbial and viral infections.
SUMMARY OF THE INVENTION
An improved assay is disclosed for detecting viral nucleic acid sequences. The improvement involves concentrating viral particles from a biological sample by centrifugation. Once concentrated, the nucleic acid molecules in the viral particles can be manipulated or detected using any suitable procedure. Many such procedures are known and can be used with the concentrated viral nucleic acid molecules. Preferably, the concentrated viral nucleic acid is detected using a detection assay. A preferred form of the disclosed assay is performed generally as follows. A test sample of blood is collected with a chemically inert device and serum is separated. The serum is subjected to a centrifugation step which concentrates the viral particles. This step has a significant effect on the ac
Chan Ming
Garcia Mariana
Lörincz Attila
Arnall Golden & Gregory LLP
Digene Corporation
Horlick Kenneth R.
Siew Jeffrey
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