Sensitive and accurate quantitation of hepatitis B virus DNA using a kinetic fluorescence detection system (TaqMan PCR).

The laboratory diagnosis of hepatitis B virus (HBV) infection is based mainly on serological assays. Yet the detection and quantitation of viral DNA is necessary when addressing directly the question of infectivity or when monitoring the viral load during therapy. Standard hybridization assays allow for exact quantitation, but their sensitivity is limited to 10(5)-10(6) viral genomes per ml of serum. The most sensitive tests for HBV DNA are nested PCR systems, which recognize virtually one molecule of the target DNA per reaction. However, these assays only provide very coarse quantitative statements. To take advantage of both methods, a new assay for HBV DNA is described based on the commercial TaqMan system. This assay is capable of quantifying HBV DNA from the theoretical lower limit up to 10(10) genome equivalents per ml of serum and, thus, covers the complete range of naturally occurring states of infections. The method was calibrated on the basis of serial plasmid dilutions and compared with a well-established nested PCR system. More than 100 HBV positive sera and serial dilutions of the Eurohep standard for both ad and ay subtypes were analyzed. The assay reliably detected all HBV positive samples. It shows minimal run-to-run deviations, allows for quantitation that covers eight orders of magnitude, and finally, completely avoids the risk of cross-contamination by PCR products. Thus, this technique combines the sensitivity of PCR amplification and the quantitation potential of hybridization tests and it is time efficient and safer.

Specific glycation of albumin depends on its half-life.

Some suggest that measurements of plasma fructosamine concentration should be corrected for the plasma protein (or albumin) concentration because the extent of glycation per volume depends on both protein and glucose concentrations. Several reports, however, demonstrate a poor correlation between plasma fructosamine and albumin concentrations in diabetic patients. In vitro kinetic and in vivo studies have shown that glycation is also dependent on the half-lives of plasma proteins. Because a decrease in plasma albumin diminishes its catabolism, we speculated that low albumin concentrations are associated with a greater extent of glycation on a molar basis (specific glycation) and vice versa. To test this hypothesis, we studied plasma albumin, total protein, and fructosamine in 63 nondiabetic subjects with normal plasma fasting glucose concentrations and hemoglobin A1c between 5.1% and 5.9%. Plasma fructosamine was poorly correlated with albumin concentration (r = 0.348) but a logarithmic plot of the specific glycation of albumin vs albumin concentration showed a better correlation (r = -0.842), suggesting that the kinetic considerations were operating in vivo. Therefore, because lower specific glycation of plasma albumin "compensates" for higher concentration, correction of fructosamine for albumin content in patients will overestimate mean blood glucose when albumin is low and underestimate it when albumin is high.