Branched DNA signal amplification for direct quantitation of nucleic acid sequences in clinical specimens

Abstract

In this chapter I have reviewed the development of bDNA as a method for quantitation of nucleic acid targets and the application of this technology to the study of infectious diseases and cell biology. The ability to quantify viral nucleic acids in clinical specimens has led to a better understanding of the pathogenesis of chronic viral infections such as HIV-1, HCV, and HBV. The information provided by these methods can also be important in the management of patients with these infections. The prognostic value of a single baseline HIV-1 RNA level rivals that surgical staging procedures for cancer, which are among the most powerfully predictive tests in medicine (Mellors et al., 1996). These methods have been used to assess rapidly the effects of antiviral therapy, which has both expedited the development of antiviral drugs and improved the management of patients with HIV-1 and HCV infections. bDNA has several characteristics that distinguish it from the quantitative target amplification systems, including better tolerance of target sequence variability, more direct measurement of target, simpler sample preparation, and less sample-to-sample variation. However, the first- and second-generation bDNA assays lacked sensitivity compared with the target amplifications systems. The changes incorporated into the third-generation assays have effectively increased the signal-to-noise ratio to such a high level that the analytical sensitivity of system 8 bDNA approaches that of PCR. In theory, bDNA can be made even more sensitive by increasing both the sample volume and the signal-to-noise ratio. Nonspecific hybridization can be further reduced by finding more effective blockers for the solid phase or by redesigning the amplifier molecule or the solid phase itself. The increased sensitivity may create new applications for the technology in filter and in situ hybridization assays.