Continuous monitoring of the polymerase chain reaction for hepatitis B virus: detection and quantitation;
Hepatitis B Virus (HBV) is a major health risk in spite of increasing diagnostic and management efforts. The polymerase chain reaction (PCR) is currently the most sensitive method of detecting HBV DNA. A donor population composed of 45 HBV negative and 45 HBV positive patients was used for this study. A microvolume fluorometer integrated with a thermal cycler combined with fluorescent methods were used to detect HBV DNA. Fluorescent intensity levels were obtained once per cycle during PCR. The double strand DNA (dsDNA) specific dye SYBR® Green I was used to monitor dsDNA production during PCR amplification. In addition, sequence specific, fluorescently labeled hybridization FRET probes were used to monitor the production of desired PCR product. Both the dsDNA dye and the hybridization FRET probes were successful in discriminating between the HBV negative samples and the HBV positive samples. When the dsDNA dye was used, any sample with a fluorescent level (background subtracted and normalized) > 0.180 at cycle number 29 was called positive (avg. positive samples = 0.470). When hybridization FRET probes were used, any sample background subtracted) with a fluorescent ratio (I-670/I-530) > 0.006 at cycle number 29 was called positive (avg. positive samples = 0.157). Two new methods for quantitation, threshold interpolation and intersect point (IP), were correlated with a hybrid capture-chemiluminescent method. The hybrid capture-chemiluminescent method does not involve PCR. The threshold interpolation method requires post-PCR analysis of fluorescent curves obtained during PCR. The user defines a fluorescent threshold from which a standard curve is established and unknown starting concentrations are calculated. The intersect point method also requires post-PCR analysis of fluorescent curves. The cycle number at which the fluorescent curve has the greatest estimated maximal slope is determined and a standard curve is calculated. The average log number of copies per |iL serum were 4.375 for SYBR® Green I (threshold), 3.638 for SYBR® Green I (IP), 4.844 for hybridization FRET probes (threshold), 5.049 for hybridization FRET probes (IP), compared to 5.017 for the Digene hybrid capture system™. The correlation coefficients for the different methods to the Digene hybrid capture system™ were 0.633 for SYBR® Green I (threshold), 0.466 for SYBR® Green I (IP), 0.367 for hybridization FRET probes (threshold), and 0.515 for hybridization FRET probes (IP).
University of Utah
Hepatitis B Virus; Polymerase Chain Reaction;
University of Utah;
Relation-Is Version Of
Digital reproduction of “Continuous monitoring of the polymerase chain reaction for hepatitis B virus: detection and quantitation”. Spencer S. Eccles Health Sciences Library.