Tandem blocking of PCR extension to form a single-stranded overhang for facile, visual, and ultrasensitive gene detection

Abstract

In order to detect a predetermined gene in a field test, a facile, visual, and ultrasensitive approach without the need of special and expensive machines is required. In this study, a gene in the Ebola virus was targeted as an example for diagnosis. The key strategy is to incorporate molecular blockers (azobenzene-bearing moieties or thymine dimers) in tandem in one of the PCR primers and stop the polymerase extension there to form a single-stranded overhang. The PCR product was added to the dispersion of gold nanoparticles which were labelled with a probe oligonucleotide. When the Ebola virus-specific gene existed in the specimen, the oligonucleotide on the gold particles formed a double-helix with the single-stranded overhang, and thus the dispersion remained red in color. In the absence of the gene, however, the dispersion rapidly turned to blue because of nanoparticle aggregation. The difference was explicit even when the initial specimen involved only 1 copy of the gene. Accordingly, “whether the patient is infected by the virus or not” can be easily and visually judged by the naked eye.