An internal amplification control for quantitative nucleic acid analysis using nanoparticle-based dipstickbiosensors

Quantitative analysis of virus nucleic acids is essential for monitoring the efficacy of medical treatment based on the copy numbers of virus's RNA or DNA in blood. To quantitatively detect virus nucleic acids in blood, here an internal amplification control (IAC) coupled with a nanoparticle-based DNA biosensor was proposed. The IACs with a specific sequence were designed and spiked into serum before nucleic acids extraction. Sequences of the IACs and the targets only differ in the base order of one PCR priming site; thus, the IACs and the targets are identical in Tm, giving the same amplification efficiency during PCR. To visually detect amplicons, a dipstick biosensor based on streptavidin-functionalized nanoparticles is employed. By comparing color densities of a test zone with an IAC zone on the biosensor, the content of the target in serum can be semi-quantitatively analyzed. This approach has achieved the detection of HBV DNA at approximately 100 copies of the pathogen load. The feasibility of this method is demonstrated by successful semi-quantification of pathogen load in 30 clinical samples from HBV-infected patients. These data indicate that the introduction of an IAC and nanoparticle-based dipstick-type biosensor could be a powerful tool in point of care testing (POCT).

► We proposed an IAC-based dipstick-type biosensor for quantifying pathogen DNA.
► Quantification was achieved by using an IAC with a sequence similar to the target.
► IAC can monitor the reliability of the whole process and avoid false negatives.
► Two-step PCR was used for quick and specific amplification.