磁纳米散射探针暗场超灵敏检测具核梭杆菌

Ultra-sensitive detection of Fusobacterium nucleatum with magnetic nanoparticle probe in dark-field

摘要:

【背景】具核梭杆菌(Fusobacterium nucleatum)作为机会致病菌，能够引起许多感染性疾病，已被证实是促直肠癌发展的潜在重要危险因素，临床检测中亟需一种快速简单检测F. nucleatum的方法。【目的】通过建立一种磁纳米探针结合暗场显微镜直接观察计数的方法，可方便快速地检测样本中具核梭杆菌的数量。【方法】在磁纳米颗粒(magnetic nanoparticle，MNP)表面修饰制备的抗F. nucleatum抗体，构建一种特异性结合F. nucleatum的MNP探针。此外，比较MNP探针-暗场显微计数法与实时荧光定量PCR (qPCR)方法检测F. nucleatum的灵敏度。【结果】该方法的检测限可低至3.42×10¹ copies/µL，比qPCR的灵敏度高5倍左右。在实际样本的检测中，该方法与qPCR方法所检测F. nucleatum数量保持一致。【结论】本研究建立的方法用于检测F. nucleatum，操作简单、检测快速(约30 min)、灵敏且成本低，有应用于临床样本检测的前景。

Abstract:

[Background] Fusobacterium nucleatum, an opportunistic pathogen causing infectious diseases, is a risk factor for the occurrence of colorectal cancer. Simple and rapid techniques are urgently needed for the detection of F. nucleatum in clinical practice. [Objective] In this study, we established a direct observation and counting method to count F. nucleatum cells in samples with magnetic nanoparticle (MNP) probe under dark-field microscopy. [Methods] We prepared the MNP probe by modifying MNPs with the homemade polyclonal antibodies against F. nucleatum, which can bind to F. nucleatum specifically. Furthermore, we compared the sensitivity of our method with that of real-time fluorescence quantitative PCR (qPCR) for detection of F. nucleatum. [Results] The method established in this study showed the limit of detection as low as 3.42×10¹ copies/μL and the sensitivity 5 times higher than that of qPCR. For detection of the real samples, the results of ounting F. nucleatum by our method are consistent with that by qPCR. [Conclusion] The established method is simple, rapid (within about 30 min), sensitive, and economical for detecting F. nucleatum, which has the potential to serve the detection of clinical samples.