Assay for the detection and quantification of HBV cccDNA by real-time PCR
First Claim
1. A method for detecting covalently closed circular cccDNA of a Hepatitis B virus HBV in the form of a HBV cccDNA genome, the method comprising the steps of:
- a) obtaining a sample of liver cells infected with HBV cccDNA virus from a patient;
b) preparing at least one primer for applying to at least one end of the HBV cccDNA virus;
c) amplifying said HBV cccDNA virus by a polymerase chain reaction (PCR) using said at least one primer;
d) preparing at least one probe for applying to said HBV cccDNA gnome, wherein said probe comprises a dye and a dye quencher; and
e) conducting a second polymerase chain reaction to bind said at least one primer to said at least one probe so that said dye and said dye quencher in said probe are separated allowing said HBV cccDNA to be detected via said dye.
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Accused Products
Abstract
The persistence of covalently closed circular (ccc) DNA of Hepatitis B Virus (HBV) in liver cells is believed to be the major reason for relapse after completion of HBV antiviral therapy. Up to now, there is no sensitive method to quantify cccDNA in infected liver cells. A set of primers were designed to specifically amplify DNA fragments from HBV cccDNA but not from viral genomic DNA. A good linear range was obtained when 100 to 107 copies of HBV cccDNA were used as template in the quantitative real-time PCR. Not only is this method rapid, economical, highly sensitive, it can be used to monitor HBV cccDNA in infected human liver biopsies and to guide patients undergoing long-term anti-HBV therapy.
5 Citations
22 Claims
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1. A method for detecting covalently closed circular cccDNA of a Hepatitis B virus HBV in the form of a HBV cccDNA genome, the method comprising the steps of:
-
a) obtaining a sample of liver cells infected with HBV cccDNA virus from a patient;
b) preparing at least one primer for applying to at least one end of the HBV cccDNA virus;
c) amplifying said HBV cccDNA virus by a polymerase chain reaction (PCR) using said at least one primer;
d) preparing at least one probe for applying to said HBV cccDNA gnome, wherein said probe comprises a dye and a dye quencher; and
e) conducting a second polymerase chain reaction to bind said at least one primer to said at least one probe so that said dye and said dye quencher in said probe are separated allowing said HBV cccDNA to be detected via said dye. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A kit for the detection of HBV cccDNA in a patient comprising:
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a) a plurality of forward amplification primers for detecting cccDNA wherein said forward primers consist of;
(5′
-(SEQ ID NO;
1) -3′
);
(5′
-(SEQ ID NO;
2) -3′
);
(5′
-(SEQ ID NO;
3)-3′
); and
b) a plurality of reverse primers wherein said reverse primers consist of;
(5′
-(SEQ ID NO;
4)-3′
);
(5′
-(SEQ ID NO;
5)-3′
);
(5′
-(SEQ ID NO;
6)-3′
). - View Dependent Claims (20, 21, 22)
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Specification