A non-optical multiplexed PCR diagnostic platform for serotype-specific detection of dengue virus

Abstract

Viable point-of-care multi-target diagnostics requires single-sample multiplexed detection in a single PCR reactor with non-optical instruments. We report an integrated multiplexed PCR chip platform with a novel ion-selective membrane sensor array that eliminates the key selectivity and sensitivity issues for multiplexed PCR, such as primer dimerization and nonspecific amplification (mis-priming). The depletion action of the ion-selective membrane renders the sensor insensitive to pH and ionic strength variation or non-specific binding, which plague the current electrochemical sensor arrays. We validate the platform with robust, sensitive and selective detection of each of the four dengue virus (DENV) serotypes, even in the presence of misprimed non-target products, with plasma samples spiked with heterogeneous RNA populations that include all the serotypes. The assay time is ∼ 90 min with a detection sensitivity of DENV RNA template down to 100 copies per mL. By replacing the optical sensing technology of RT-PCR with the membrane sensor, the platform is scalable and can allow simultaneous screening for multiple viral infections in a single sample within a single PCR reactor chip for point-of-care applications.