Inoue Shinnosuke, Becker Annie L, Kim Jong-Hoon, Shu Zhiquan, Soelberg Scott D, Weigel Kris M, Hiraiwa Morgan, Cairns Andrew, Lee Hyun-Boo, Furlong Clement E, Oh Kieseok, Lee Kyong-Hoon, Gao Dayong, Chung Jae-Hyun, Cangelosi Gerard A
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, United States of America.
PLoS One. 2014 Jan 22;9(1):e86018. doi: 10.1371/journal.pone.0086018. eCollection 2014.
An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10(6)-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.
开发了一种职业安全（生物安全）的痰液液化方案，用于基于抗体的半自动微尖端免疫荧光传感器。该方案能有效液化痰液并灭活包括结核分枝杆菌在内的微生物，同时保留结核分枝杆菌细胞表面抗原的抗体结合活性。痰液采用协同化学 - 热方案处理，即在60°C下用中等浓度的NaOH和去污剂处理5至10分钟。接种结核分枝杆菌复合体细胞的样本在处理后显示病原体的灭活程度约为10(6)倍。通过微尖端免疫传感器分析确定，处理后抗体结合得以保留。在30分钟的样本到结果过程中，该传感器能正确区分结核分枝杆菌菌种与生物安全处理痰液中天然存在的其他细胞类型，对结核分枝杆菌的检测限为100 CFU/mL。微尖端设备也是半自动的，并且已证明与低成本的LED供电荧光显微镜兼容。该设备和生物安全痰液液化方法为在实验室基础设施有限的环境中快速检测结核病打开了大门。
