The HBV DNA Quantitation Real-Time PCR kit coded HBVDNAQT.CE is intended for quantitative detection of Hepatitis B Virus DNA in human plasma with a simultaneous control of the amplification reaction through an Internal Control (IC).
The kit is intended for use in conjunction with clinical observation and laboratory markers as an indicator of disease prognosis and for use as an aid in assessing viral response to antiviral treatment.
The kit has been adapted for the use on the Real-Time Thermacyclers ABI 7500 Sequence Detection System® (Applied Biosystems™*) software SDS 1.3.1 or MX3000P® software MxPro 4.01(Stratagene™*** ).
NTRODUCTION
The hepatitis B virus is a virus belonging to the Hepadnavirus family. Its genome is a small circular, incomplete ds DNA that became a complete and super-coiled genome (cccDNA) when the virus get into the host hepatocytes. Recently eight genotypes are been identified (A through H). The virus has a high speed replicative and high frequency of mutation. This mutations lead to accumulation in each infected individual of multiple genetic variant called quasispecies. Growing evidence shows that the natural history and treatment response may differ depending on the infecting HBV genotype. However the real correlation between genotype and outcome of disease are not completely understood. Hepatitis B virus infection is a global public health problem concerning 350 million people worldwide. It can evolve to chronicity (CHB) and can be the cause of liver disease or hepatocellular carcinoma (HCC). At the present time some observation showed that the severity of liver injury during the infection, is modulated by the strength of host immune response. According on what It is possible to recognize two type of infection: acute and chronic.
In acute HBV infection hepatitis B surface antigen (HBsAg), core IgM antigen (HBcIgM), e antigen (HBeAg) are detectable in serum but ALT do not increase until the infection is well established. Levels of HBV DNA are generally very high ranging from 200 million UI/ml to 200 billion UI/ml.
RINCIPLE OF THE TEST
The HBVDNAQT.CE Kit uses the Real Time PCR technology to detected and quantify HBV DNA in the clinical plasma samples.
The specificity of the assay is first ensured by the selection of specific primers and probes as well as the selection of stringent reaction conditions.
HBV DNA, recovered from the plasma sample is extracted and amplified together with the unrelated sequence (IC) introduced into the each specimen at the beginning of sample preparation. This IC serves to demonstrate that the whole process proceeds properly for each one.
The amplified product is detected and quantified, against the standard curve, using a fluorescent reporter dye probe specific for HBV sequence. The construction of standard curve allow the determination of the viral load.
The assay is standardized against the World Health organization (WHO) 2nd International Standard for Hepatitis B virus (NIBSC Code 97/750).
Results are reported in Units per millilitre (UI/ml) or copies/ml.
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