Amplification free detection of Herpes Simplex Virus DNA

Abstract

Amplification-free detection of nucleic acids in complex biological samples is an important technology for clinical diagnostics, especially in the case where the detection is quantitative and highly sensitive. Here we present the detection of a synthetic DNA sequence from Herpes Simplex Virus-1 within swine cerebrospinal fluid (CSF), using a sandwich-like, magnetic nanoparticle pull-down assay. Magnetic nanoparticles and fluorescent polystyrene nanoparticles were both modified with DNA probes, able to hybridise either end of the target DNA, forming the sandwich-like complex which can be captured magnetically and detected by fluorescence. The concentration of the target DNA was determined by counting individual and aggregated fluorescent nanoparticles on a planar glass surface within a fluidic chamber. DNA probe coupling for both nanoparticles was optimized. Polystyrene reporter nanoparticles that had been modified with amine terminated DNA probes were also treated with amine terminated polyethylene glycol, in order to reduce non-specific aggregation and target independent adhesion to the magnetic particles. This way, a limit of detection for the target DNA of 0.8 pM and 1 pM could be achieved for hybridisation buffer and CSF respectively, corresponding to 0.072 and 0.090 femtomoles of target DNA, in a volume of 0.090 mL.