Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-12-04
2003-10-21
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S005000, C536S024300, C536S024320, C536S024330, C536S025320
Reexamination Certificate
active
06635428
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to compositions and methods for detecting hepatitis B viral nucleic acids in a test sample.
BACKGROUND OF THE INVENTION
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
One of the major causes of hepatitis are specific hepatitis viruses. There are at least six different viruses responsible for various types of hepatitis. Among them, hepatitis B virus (HBV) is the most thoroughly characterized and complex etiologic agent. The infective Dane particle consists of a viral core plus an outer surface coat. The core contains circular double-stranded DNA and DNA polymerase, and it replicated within the nuclei of infected hepatocytes. Surface coat is added in the cytoplasm and is produced in great excess; it can be detected in serum by immunologic means as hepatitis B surface antigen (HBsAg).
At least three distinct antigen-antibody systems are intimately related to HBV:
1. HBsAg is associated with the viral surface coat; its presence in serum is usually the first evidence of acute HBV infection and implies infectivity of the blood. HBsAg characteristically appears during the incubation period, usually 1 to 6 weeks before clinical or biochemical illness develops, and disappears during convalescence. The corresponding protective antibody (anti-HBs) appears weeks or months later, after clinical recovery, and usually persists for life; thus, its detection indicates past HBV infection and relative immunity;
2. Core antigen (HBcAg) is associated with the viral core. It can be found in infected liver cells but is not detectable in serum except by special techniques that disrupt the Dane particle. Antibody to HBcAg (anti-HBc) generally appears at the onset of clinical illness; thereafter, titers gradually diminish, usually for years or life. Its presence with anti-HBs is not significant beyond indicating previous HBV infection. It is also regularly found in chronic HBsAg carriers, who do not mount an anti-HBs response. In chronic infection, anti-HBc is mainly of the IgG class, whereas in acute infection, IgM anti-HBc predominates. Occasionally, IgM anti-HBc is the only marker of recent HBV infection, reflecting a “window” between disappearance of HBsAg and appearance of anti-HBs;
3. The e antigen (HBeAg) appears to be a peptide derived from the viral core. Found only in HBsAg-positive serum, HBeAg tends to parallel the production of viral DNA polymerase. Its presence, therefore, reflects more active viral replication and is generally associated with greater infectivity of the blood and a greater likelihood of progression to chronic liver disease. In contrast, presence of the corresponding antibody (anti-HBe) points to relatively lower infectivity and usually portends a benign outcome.
HBV is often transmitted parenterally, typically by contaminated blood or blood products. Routine screening of donor blood for HBsAg can dramatically diminish post-transfusion HBV infection. Hepatitis B virus (HBV) infects approximately 400 million persons worldwide. Chronic HBV carriers provide a world-wide reservoir of infection. Prevalence varies widely according to several factor, including geography. Vertical transmission from mother to infant is partly responsible, especially where prevalence is high.
HBV is associated with a wide spectrum of liver diseases, from a subclinical carrier state to acute hepatitis, chronic hapatitis, cirrhosis, and hepatocellular carcinoma (Loeb et al. Hepatology 32: 626-629, 2000). It also has a poorly understood association with several primarily nonhepatic disorders, including polyarteritis nodosa, and other collagen vascular diseases, etc. The pathogenic role of HBV in these disorders is not clear, but in some patients there is tissue deposition of immune complex containing viral antigen.
Hepatitis B is specifically diagnosed by identifying HBsAg in serum, with or without concomitant anti-HBc. Failure to detect HBsAg does not entirely exclude hepatitis B because antigenemia may be transient. Moreover, the presence of hepatitis B virus surface antigen (HBsAg) in serum or plasma indicates HBV infection, but the detection of HBsAg does not provide information on the replicative activity of the virus. Traditionally, the secretory version of the HBV core protein (HBeAg) serves as a marker for active viral replication. In the treatment of chronic hepatitis B, the presence or absence of HBeAg is assumed to represent a high or low replicative state of HBV, respectively. However, precore mutant HBVs which do not produce HBeAg, irrespective of their rate of replication, have been described (Zaaijer et al. J. Clin. Microbiol. 32: 2088-2091, 1994).
The measurement of HBV DNA in serum has become an important tool to identify individuals with high viral replication, to monitor patients on therapy, and to predict whether antiviral therapy will be successful. With the introduction of new antivirals like lamivudine [(−)2′3′-dideoxy-3′-thiacytidine], close monitoring of patients has become increasingly important due to the occurrence of antiviral drug-resistant virus strains or the presence of flares after withdrawal from antiviral therapy (Pas et al. J. Clin. Microbiol. 38: 2897-2901, 2000; Honkoop et al. J. Hepatology 26: 1393-1395, 1997).
Several tests have been employed to detect HBV in serum and other body fluids. Immunological tests are performed by demonstration of viral antigens (HBsAg, HBcAg and HBeAg), or their respective antibodies in serum.
Hybridization techniques have also been used. Generally, such techniques involve extracting DNA from cell scrapes or biopsy materials and immobilizing it on a solid phase either directly as total DNA or as restriction fragments after resolution by gel electrophoresis. The immobilized DNA is detected most commonly by a nucleic acid probe carrying a radioactive label. To increase the sensitivity of such assays, viral nucleic acid sequences can be amplified by using, for example, the polymerase chain reaction (PCR). The products thus obtained can be identified by using conventional hybridization techniques for identification of virus types, such as Southern blotting (C. Oste, BioTechniques 6: 163, 1988; K. B. Mullis, U.S. Pat. No. 4,683,202). PCR is described in U.S. Pat. No. 4,683,195 and 4,683,202 and has been utilized to improve the sensitivity of standard hybridization methods. U.S. Pat. No. 4,562,159 discloses a method and kit which use PCR to specifically detect HBV DNA in a test sample.
Several reports disclose assays of patient samples following a nucleic acid amplification step, such as PCR (Kaneko et al., Proc. Natl. Sci. U.S.A. 86: 312-316, 1989; Larzul et al., J. Virol. Meth. 20: 227-237; Sumazaki et al., J. Med. Virol. 27: 304-308, 1989; and Theilman et al., Liver 9: 322-328, 1989). Other relevant references describe amplification primers and detection probes for human HBV (Seelig et al., DeutschMed Wochenschr 115: 1307-1312, 1990; Brunetto et al., Proc. Natl. Acad. Sci., USA 88: 4186-4190, 1991; Brunetto et al., Prog. Clin. Biol. Res. (U.S.) 364: 211-216, 1991; Fiordalisi et al., J. Med. Virol. 31: 297-300, 1990; Liang et al., Hepatology 12(2): 204-212, 1990; Lo et al., J. Clin. Microbiol. 28(6): 1411-1416, 1990; Pasquinelli et al., J. Med. Virol 31: 135-140, 1990; Musso, PCT/US88/03735; Urdea, PCT/US90/02049; Urdea, et al., Gene 61: 253-264, 1987; and Urdea, PCT/US91/00213). The method and hybridization assays using self-quenching fluorescence probes with and/or without internal controls for detection of nucleic acid application products are known in the art, for example, U.S. Pat. Nos. 6,258,569; 6,030,787; 5,952,202; 5,876,930; 5,866,336; 5,736,333; 5,723,591; 5,691,146; and 5,538,848. Moreover, publications for detection of HBV using Real-time PCR (Taqman systems) include the following: Chen et al., J. Med. Virol. 65(2): 250-6, 2001; Meng et al., J. Clin Microbiol. 39(8): 29
Hamdan Hasnah
Lewinski Michael
Pasupuleti Vijaya
Foley & Lardner
Hill Myron G.
Housel James
Quest Diagnostics Investments Incorporated
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