Bioluminescence DNA hybridization assay for Plasmodium falciparum based on the photoprotein aequorin
Bioluminescence DNA hybridization assay for Plasmodium falciparum based on the photoprotein aequorin
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DOI：
10.1021/ac0702847
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发表时间：
2007-06-01
影响因子：
7.4
通讯作者：
Daunert, Sylvia
中科院分区：
文献类型：
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作者：
Doleman, Leslie;Davies, Logan;Daunert, Sylvia
利用发光蛋白水母发光蛋白作为生物发光标记，建立了一种检测恶性疟原虫的生物发光DNA杂交方法。目前检测疟疾的金标准是光学显微镜，其可以检测到低至约50个寄生虫/μ L血液，但具有低通量、高成本和高技能要求，这限制了该方法的适用性，特别是在最需要疟疾检测的发展中地区。利用水母发光蛋白作为生物发光标记物提供了高信噪比和低至阿托摩尔水平的可靠检测的优点，从而允许开发高灵敏度和小型化的高通量生物发光测定。本研究利用链霉亲和素-水母发光蛋白与靶DNA竞争杂交的原理，建立了一种检测恶性疟原虫的DNA杂交方法。该生物发光杂交试验证明检测限为3 pg/mu L，并用于检测标准和加标人血清样品中的靶DNA。DNA杂交试验是在微孔板的格式，而不需要样品PCR扩增，显示了这种方法在平行分析样品的低培训人员，如通常在发展中地区遇到的潜在适用性。
A bioluminescence DNA hybridization assay for the detection of 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 similar to 50 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.