Mido Tomotaka, Schaffer Eric M, Dorsey Robert W, Sozhamannan Shanmuga, Hofmann E Randal
CBRN Detection Technology Section, CBRN Defense Technology Division, Advanced Defense Technology Center, Acquisition, Technology and Logistics Agency (ATLA), Tokyo, Japan.
Leidos, Inc., Aberdeen Proving Ground, MD, USA; Biosciences Division, U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Grounds, Edgewood, MD, USA.
J Microbiol Methods. 2018 Sep;152:143-147. doi: 10.1016/j.mimet.2018.07.022. Epub 2018 Aug 2.
Phages are natural predators of bacteria and have been exploited in bacterial detection because of their exquisite specificity to their cognate bacterial hosts. In this study, we present a "proof of concept" bacteriophage amplification-coupled assay as a surrogate for detecting a bacterium present in a sample. The assay entails detection of progeny phage resulting from infection and subsequent growth inside the bacterium present in suspected samples. This approach reduces testing time and enhances sensitivity to identify pathogens compared to traditional overnight plaque assay. Further, the assay has the ability to discriminate between live and dead cells since phages require live host cells to infect and replicate. To demonstrate its utility, phage MS2 amplification-coupled, bead-based sandwich type immunoassay on the Luminex® MAGPIX instrument for Escherichia coli detection was performed. The assay not only showed live cell discrimination ability but also a limit of E. coli detection of 1 × 10 cells/mL of live cells after a 3-h incubation. In addition, the sensitivity of the assay was not impaired in the presence of dead cells. These results demonstrate that bacteriophage amplification-coupled assay can be a rapid live cell detection assay compared to traditional culture methods and a promising tool for quick validation of bacterial inactivation. Combined with the unique multiplex bead chemistry of the Luminex® MAGPIX platform, the phage assay can be expanded to be an ultra-deep multiplex assay for the simultaneous detection of multiple pathogens using specific phages directed against the target pathogens.
噬菌体是细菌的天然捕食者，由于其对同源细菌宿主具有极高的特异性，已被用于细菌检测。在本研究中，我们提出了一种“概念验证”噬菌体扩增耦合检测方法，作为检测样品中存在细菌的替代方法。该检测方法需要检测由感染以及随后在疑似样品中存在的细菌内生长所产生的子代噬菌体。与传统的过夜蚀斑检测相比，这种方法减少了检测时间，并提高了识别病原体的灵敏度。此外，由于噬菌体需要活的宿主细胞来感染和复制，该检测方法有能力区分活细胞和死细胞。为了证明其效用，我们在Luminex® MAGPIX仪器上进行了基于磁珠的夹心型免疫分析，耦合噬菌体MS2扩增以检测大肠杆菌。该检测方法不仅显示出活细胞区分能力，而且在3小时孵育后，对活细胞的大肠杆菌检测限为1×10个细胞/毫升。此外，在存在死细胞的情况下，该检测方法的灵敏度并未受损。这些结果表明，与传统培养方法相比，噬菌体扩增耦合检测方法可以成为一种快速的活细胞检测方法，并且是用于快速验证细菌灭活的有前景的工具。结合Luminex® MAGPIX平台独特的多重磁珠化学技术，噬菌体检测方法可以扩展为一种超深度多重检测方法，使用针对目标病原体的特异性噬菌体同时检测多种病原体。
