Okanojo Masahiro, Miyashita Noe, Tazaki Aya, Tada Hiroko, Hamazoto Fumiaki, Hisamatsu Mitsuko, Noda Hideyuki
Central Research Laboratory, Hitachi Ltd, Tokyo, Japan.
Infrastructure Systems Company, Hitachi Ltd, Tokyo, Japan.
Luminescence. 2017 Aug;32(5):751-756. doi: 10.1002/bio.3246. Epub 2016 Dec 13.
We have developed an automated high-sensitive ATP bioluminometer for detecting single bacterium. The apparatus consists of a tube rack for setting reagents and samples, two washing baths for preventing sample carry-over from dispenser nozzle, and x-, y-, z- actuators for moving the dispenser, and an high-sensitive optical system. The reaction tube was selected to reduce the background signal intensities for the ATP bioluminescence measurement. The background signal intensity of the reaction tube was 18 RLU, which is almost the same as the dark counts of the photomultiplier (16 RLU). The ATP calibration curve was linear from 0 to 5 amol (its slope = 22.4 RLU/amol and 3.3 SD of the blank sample signal = 17.9 RLU), and the detection limit of 0.8 amol was obtained. The relationship between intracellular ATP and CFU in Escherichia coli (ATCC25922) was kept linearity from 0 to 20 CFU, and the intracellular ATP (amol) per CFU was calculated to be 3.3 amol/CFU (R2 = 0.9713). Moreover, the relationship between intracellular ATP and CFU in Staphylococcus aureus (ATCC25923) was also kept linearity from 0 to 30 CFU, and the amol/CFU was calculated to be 1.6 amol/CFU (R2 = 0.9847). The automated ATP bioluminometer has ultra-high sensitivity and will be a powerful tool for measuring ATP luminescence derived from small number of bacteria.
我们开发了一种用于检测单个细菌的自动化高灵敏度ATP生物发光计。该仪器由一个用于放置试剂和样品的试管架、两个用于防止样品从分配器喷嘴残留的清洗槽、用于移动分配器的x、y、z轴驱动器以及一个高灵敏度光学系统组成。选择反应管以降低ATP生物发光测量的背景信号强度。反应管的背景信号强度为18 RLU，几乎与光电倍增管的暗计数（16 RLU）相同。ATP校准曲线在0至5 amol范围内呈线性（其斜率 = 22.4 RLU/amol，空白样品信号的3.3倍标准差 = 17.9 RLU），检测限为0.8 amol。大肠杆菌（ATCC25922）中细胞内ATP与CFU之间的关系在0至20 CFU范围内保持线性，计算得出每CFU的细胞内ATP（amol）为3.3 amol/CFU（R2 = 0.9713）。此外，金黄色葡萄球菌（ATCC25923）中细胞内ATP与CFU之间的关系在0至30 CFU范围内也保持线性，计算得出amol/CFU为1.6 amol/CFU（R2 = 0.9847）。这种自动化ATP生物发光计具有超高灵敏度，将成为测量少量细菌产生的ATP发光的有力工具。
