Hbv Dna Detection and Quantitation

The commercially available tests for quantitation of HBV DNA in serum and plasma are listed in Table 37-2. These tests employ either signal or target amplification. All of these assays are available as research-use-only kits or analyte-specific reagents, since the Food and Drug Administration (FDA) has not cleared any of these reagents for diagnostic use. In addition, a number of laboratory-developed conventional and real-time polymerase chain reaction (PCR) tests have been described.4,13-15 All of the quantitative HBV DNA test formats have been used in monitoring the status of HBV infection before and after treatment.

The Hybrid Capture tests (Digene, Gaithersburg, MD) are liquid hybridization assays employing full-length genomic RNA probes and antibodies that bind to DNA-RNA hybrids to quantitate HBV DNA in serum and plasma specimens. The dynamic ranges of the Standard and Ultrasensitive Hybrid Capture II assays are 200,000 to 2 billion genome copies/ml and 5,000 to 60 million genome copies/ml, respectively.

The Versant HBV assay (Bayer Corp, Tarrytown, NY) uses branched DNA (bDNA) technology for HBV DNA quantitation. The virus is concentrated from 1 ml of serum by ultracentrifugation to achieve greater sensitivity. Quantitation of HBV DNA in clinical samples is determined from a standard curve generated with each run using a set of HBV DNA standards included as part of the kit. The Versant HBV assay has a dynamic range of 700,000 to 5 billion copies/ml.

The Amplicor HBV Monitor Test (Roche Diagnostics, Indiannapolis, IN) uses PCR for HBV DNA quantitation16 and has the greatest sensitivity of all the available assays, detecting as few as 200 HBV genome copies/ml. The PCR primers are biotin labeled and target conserved sequences in the precore/core region of the HBV genome. A known number of copies of a quantitation standard (QS) are added to each specimen aliquot prior to DNA isolation. The QS is a DNA fragment with 5' and 3' sequences that are complementary to the HBV PCR primers with a unique internal sequence. After PCR amplification, the HBV and QS amplicons are detected using specific probes for internal sequences of the target and QS in a solid phase assay with an avidin-enzyme conjugate and a colorimetric substrate. HBV DNA in the serum sample is quantitated by reference to the QS signal. The amplification, hybridization,

Table 37-2. Commercially Available HBV DNA Tests

Test Kit

Method

Manufacturer

Gene Target

Dynamic Range*

Standard Hybrid Capture II

Ultra-Sensitive Hybrid Capture II

Versant HBV Amplicor HBV Monitor

TaqMan HBV Analyte-Specific Reagent

Liquid hybridization

Liquid hybridization

Branched DNA PCR

Real-time PCR

Digene (Gaithersburg, MD)

Digene (Gaithersburg, MD)

Bayer Corp (Tarrytown, NY) Roche Diagnostics

(Indiannapolis, IN) Roche Diagnostics (Indiannapolis, IN)

Full-length RNA probet Full-length RNA probet Multiple probes Precore/core

2 x lO2 to 2 x lO7

* Genome copies/ml. t Genotypes A and D.

and detection steps can be performed using the COBAS Amplicor analyzer.17

A real-time PCR assay for HBV DNA based on TaqMan chemistry is available from Roche Diagnostics as an analyte-specific reagent (ASR). This assay combines the analytical sensitivity of conventional PCR with the broad dynamic range of the Hybrid Capture and Versant bDNA tests, and simplifies the analytical process relative to the PCR tests by combining the amplification and detection steps.

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