Amplification and non-isotopic detection of specific DNA sequences in a single microtitre well

Abstract

We report the development of a convenient and reliable polymerase chain reaction (PCR)-based microassay for the amplification and detection of specific DNA sequences with potential applications in the diagnostics field. The major features of our system are: (a) the complete system is carried out entirely in the same microtitre well; (b) the PCR is performed in two instead of the traditional three temperatures, thus reducing the time for 35 cycles to under 2 h; (c) the probe is already immobilized onto the solid phase, allowing direct hybridization of the PCR products; (d) one of the two primers is already biotinylated at the 5' end, thus detecting one of the two actual specific products, and (e) the whole process is designed to an enzyme-linked immunosorbent assay (ELISA)-like system for easy use and takes only 3 h, rendering the system particularly suitable for a busy clinical laboratory and automation. The method was successfully applied for the detection of human immunodeficiency virus type 1 (HIV-1) from patient lymphocyte samples.