We have developed an HBV DNA detection system that uses the TMA-HPA technologies (12
). The advantages of the TMA-HPA method include isothermal amplification and nonradioactive, single-step differentiation of hybridized and unhybridized probe. The assay procedure involves sample and reagent additions and temperature incubations, but it is not technically demanding. In a moderately sized run (80 specimens and standards), the sample addition and the assay steps can be completed in 1 and 4 h, respectively, when they are done manually. It is possible to incorporate this assay into the normal work routine of clinical laboratories.
Results obtained by serially diluting the HBV DNA standard indicated that our assay has a broad dynamic range of from 3.7 to 8.7 LGE/ml (5 logs) and good quantitative linearity. The detection range of the assay was expanded by using two probe solutions with two different specific activities for the detection of low and high amplicon concentrations. When HBV DNA was extracted from the serum samples by an appropriate method, such as the phenol-chloroform method, guanidine method, or NaI method, and the extracted DNA was used as the sample, it was possible to increase the sensitivity of the assay, and the assay still possessed linearity beyond the current lower limit of 3.7 LGE/ml (data not shown).
Several groups have reported the development of assay systems for the detection of HBV DNA with or without DNA amplification (2
). The assay systems without amplification had lower detection sensitivities (from about 6.0 to 9.0 LGE/ml) and high quantitative accuracies, while the assay systems with amplification had higher sensitivities but lower quantitative accuracies. The detection ranges of both of these assay systems were apparently about 3 logs, regardless of whether amplification was used. As shown from the clinical performance of the assay in Fig. , HBV DNA amounts in patients with various disease conditions were widely distributed over more than 5 logs. Furthermore, even HBV DNA levels in a single patient varied markedly (Fig. ). Two clinical studies have suggested that HBV DNA amounts differ markedly in hepatitis B patients and carriers and that the detection range of some assays was apparently too narrow to monitor the amount of HBV DNA (11
). The results reported here suggest that diagnosis of HBV infection and monitoring of patients with HBV infection may require a test not only with adequate sensitivity but also one with a very wide detection range. The quantitative test for the detection of HBV that we have developed has adequate sensitivity and a broad dynamic range for monitoring the condition and prognosis of HBV patients, carriers, and especially, patients undergoing interferon therapy.