Gabriel Shobana, Rasheed Abdul Khaliq, Siddiqui Ruqaiyyah, Appaturi Jimmy Nelson, Fen Leo Bey, Khan Naveed Ahmed
Department of Biological Sciences, School of Science and Technology, Sunway University, 47500, Bandar Sunway, Selangor, Malaysia.
Research Centre for Nano-Materials and Energy Technology, School of Science and Technology, Sunway University, Bandar Sunway, Malaysia.
Parasitol Res. 2018 Jun;117(6):1801-1811. doi: 10.1007/s00436-018-5864-0. Epub 2018 Apr 20.
Brain-eating amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) have gained increasing attention owing to their capacity to produce severe human and animal infections involving the brain. Early detection is a pre-requisite in successful prognosis. Here, we developed a nanoPCR assay for the rapid detection of brain-eating amoebae using various nanoparticles. Graphene oxide, copper and alumina nanoparticles used in this study were characterized using Raman spectroscopy measurements through excitation with a He-Ne laser, while powder X-ray diffraction patterns were taken on a PANanalytical, X'Pert HighScore diffractometer and the morphology of the materials was confirmed using high-resolution transmission electron microscopy (HRTEM). Using nanoparticle-assisted PCR, the results revealed that graphene oxide, copper oxide and alumina nanoparticles significantly enhanced PCR efficiency in the detection of pathogenic free-living amoebae using genus-specific probes. The optimal concentration of graphene oxide, copper oxide and alumina nanoparticles for Acanthamoeba spp. was determined at 0.4, 0.04 and 0.4 μg per mL respectively. For B. mandrillaris, the optimal concentration was determined at 0.4 μg per mL for graphene oxide, copper oxide and alumina nanoparticles, and for Naegleria, the optimal concentration was 0.04, 4.0 and 0.04 μg per mL respectively. Moreover, combinations of these nanoparticles proved to further enhance PCR efficiency. The addition of metal oxide nanoparticles leads to excellent surface effect, while thermal conductivity property of the nanoparticles enhances PCR productivity. These findings suggest that nanoPCR assay has tremendous potential in the clinical diagnosis of parasitic infections as well as for studying epidemiology and pathology and environmental monitoring of other microbes.
食脑变形虫（棘阿米巴属、曼氏巴贝斯虫、福氏耐格里阿米巴）因其能够引发涉及大脑的严重人类和动物感染而受到越来越多的关注。早期检测是成功预后的先决条件。在此，我们开发了一种纳米PCR检测方法，用于使用各种纳米颗粒快速检测食脑变形虫。本研究中使用的氧化石墨烯、铜和氧化铝纳米颗粒通过氦氖激光激发的拉曼光谱测量进行表征，同时在帕纳分析公司的X'Pert HighScore衍射仪上获取粉末X射线衍射图谱，并使用高分辨率透射电子显微镜（HRTEM）确认材料的形态。使用纳米颗粒辅助PCR，结果表明，氧化石墨烯、氧化铜和氧化铝纳米颗粒在使用属特异性探针检测致病性自由生活变形虫时显著提高了PCR效率。棘阿米巴属的氧化石墨烯、氧化铜和氧化铝纳米颗粒的最佳浓度分别确定为每毫升0.4、0.04和0.4微克。对于曼氏巴贝斯虫，氧化石墨烯、氧化铜和氧化铝纳米颗粒的最佳浓度确定为每毫升0.4微克，对于耐格里阿米巴，最佳浓度分别为每毫升0.04、4.0和0.04微克。此外，这些纳米颗粒的组合被证明能进一步提高PCR效率。金属氧化物纳米颗粒的添加导致优异的表面效应，而纳米颗粒的热传导性能提高了PCR产量。这些发现表明，纳米PCR检测方法在寄生虫感染的临床诊断以及研究其他微生物的流行病学、病理学和环境监测方面具有巨大潜力。
