Department of Biological Sciences, Purdue University, West Lafayette, Indiana.
Purdue University Interdisciplinary Life Science Program (PULSe), West Lafayette, Indiana.
PLoS One. 2021 Apr 6;16(4):e0249473. doi: 10.1371/journal.pone.0249473. eCollection 2021.
Due to the nascency of bacteriophage-based pathogen detection technologies, several practical hurdles stand in the way between providing promising proof-of-concept data and development of robust detection platforms. One such hurdle, and the focus of this work, is the development of methods for transitioning laboratory stocks of bacteriophage into functional, consistent, and shelf-stable delivery methods in commercial detection kits. Research described here was undertaken to evaluate two methods for their ability to store the bacteriophage ɸV10 at ambient temperature without aqueous storage solutions while limiting loss of viability. ɸV10 is a temperate bacteriophage which solely infects the zero-tolerance food adulterant Escherichia coli O157:H7 and has been genetically modified to generate a detectable phenotype in host cells. In order to integrate this reporter bacteriophage into food-borne pathogen detection methodologies, two methods of processing phage suspensions for long-term, ambient storage were evaluated: printing solutions onto pieces of dissolvable paper and lyophilizing suspensions with sucrose. Applying phage to dissolvable paper yielded key attributes to consider when addressing phage viability, however, optimized methodology still resulted in an approximate five-log reduction in titer of viable phage. Lyophilization of ɸV10 with various concentrations of the cryoprotectant molecule, sucrose, yielded losses of approximately 0.3-log after 120 days of storage at 23°C. Liquid storage buffer samples with and without sucrose saw a reduction of viable phage of at least 3.9-log in the same period. Additionally, the ability for ɸV10 to form lysogens in an E. coli O157:H7 host was not negatively affected by lyophilization. Drying ɸV10 at ambient temperature drastically reduces the viability of the phage. However, lyophilizing ɸV10 in the presence of sucrose is an effective method for dehydration and storage of the phage in ambient environmental conditions for an extended time lending to commercial application and integration into foodborne pathogen detection methodologies.
由于噬菌体病原体检测技术还处于起步阶段，在将有前景的概念验证数据转化为稳健的检测平台方面，存在一些实际的障碍。其中一个障碍，也是本工作的重点，是开发将噬菌体的实验室库存转化为商业检测试剂盒中功能一致、稳定且易于储存的方法。这里描述的研究旨在评估两种方法在不使用水性储存溶液的情况下，在环境温度下储存噬菌体 ɸV10 的能力，同时限制其生存能力的损失。ɸV10 是一种温和噬菌体，只能感染零容忍的食品掺杂物大肠杆菌 O157:H7，并已被遗传修饰以在宿主细胞中产生可检测的表型。为了将这种报告噬菌体整合到食源性病原体检测方法中，评估了两种用于长期环境储存的噬菌体悬浮液处理方法：将噬菌体溶液打印到可溶解的纸上和用蔗糖冻干悬浮液。将噬菌体应用于可溶解的纸上，产生了在解决噬菌体生存能力时需要考虑的关键属性，然而，优化的方法仍然导致存活噬菌体的效价降低约五个对数。用不同浓度的冷冻保护剂蔗糖冻干 ɸV10，在 23°C 下储存 120 天后，效价损失约为 0.3 个对数。在同一时期，含有和不含有蔗糖的液体储存缓冲液样品中存活噬菌体的减少量至少为 3.9 个对数。此外，ɸV10 在大肠杆菌 O157:H7 宿主中形成溶原菌的能力不受冻干的影响。在环境温度下干燥 ɸV10 会大大降低噬菌体的生存能力。然而，在存在蔗糖的情况下冻干 ɸV10 是一种有效的脱水方法，可在环境条件下长时间储存噬菌体，有利于商业应用和整合到食源性病原体检测方法中。
