Bioluminescence DNA Hybridization Assay for Plasmodium falciparum Based on the Photoprotein Aequorin

摘要

A bioluminescence DNA hybridization assay for the detectionof Plasmodium falciparum, the most deadly species of malaria, using the photoprotein aequorin as a bioluminescent label has been developed. The current gold standard for the detection of malaria is light microscopy, which can detect down to approx50 parasites/(mu)L of blood, but has low-throughput, high costs, and requires high skill, which limit the applicability of the method, especially in the developing regions where malaria detection is mostly needed. The utilization of aequorin as a bioluminescence label offers the advantages of high signal-to-noise ratio and reliable detection down to attomole levels, allowing for the development of highly sensitive and miniaturized high-throughput bioluminescence assays. Herein, we developed a DNA hybridization assay for the detection of P. falciparum based on the competition between the target DNA and the signal generating DNA streptavidin-aequorin for hybridization with the probe DNA. This bioluminescence hybridization assay demonstrated a detection limit of 3 pg/(mu)L and was employed for the detection of target DNA in standard and spiked human serum samples. The DNA hybridization assay was developed in a microplate format without the need for sample PCR amplification, showing the potential suitability of this method in the parallel analysis of samples by low-trained personnel, such as that typically encountered in developing regions.