Rapid enumeration of phage in monodisperse emulsions.
Rapid enumeration of phage in monodisperse emulsions.
复制标题
单分散乳液中噬菌体的快速计数。
作者：
Katrina F. Tjhung;Sean Burnham;H. Anany;M. Griffiths;R. Derda
已经开发了基于噬菌体的检测测定用于检测活细菌，用于临床诊断、水质监测和食品安全。这些测定中的大多数以被感兴趣的细菌宿主感染的新产生的子代的形式递送阳性读数。子代细菌在充满琼脂的培养皿上通常被视为菌苔中的斑块或孔;然而，这一限速步骤需要长达12小时的孵育时间。我们先前已经描述了悬浮在全氟化油中的培养基液滴内噬菌体M13的扩增;液滴中的单个噬菌体M13在3-4小时内产生10（7）个拷贝。在这里，我们描述了在单分散液滴中包封报告基因，裂解性T4-LacZ和非裂解性M13，也可以用于噬菌体的快速计数。液滴中的区室化加速了来自报告酶的信号的发展;对“阳性”液滴的计数产生范围为10（2）至10（6）pfu/mL的噬菌体颗粒的准确计数。对于T4-LacZ噬菌体的计数，荧光信号在短至90分钟内出现。与批量测定不同，乳液中的定量是稳健的并且对环境条件的波动不敏感（例如，温度）。在没有注射泵和便携式荧光成像解决方案的情况下，使用重力驱动流的无动力乳化使得该技术有望在低资源环境中的护理点使用。这种基于液滴的噬菌体计数方法可以加速和简化已经开发了报告噬菌体的病原体的即时检测。
Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed.
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影响因子：
16.6
作者：
Derda, Ratmir;Tang, Sindy K. Y.;Whitesides, George M.
通讯作者：
Whitesides, George M.
影响因子：
7.4
作者：
Selck, David A.;Karymov, Mikhail A.;Sun, Bing;Ismagilov, Rustem F.
通讯作者：
Ismagilov, Rustem F.
影响因子：
46.2
作者：
Vincent ME;Liu W;Haney EB;Ismagilov RF
通讯作者：
Ismagilov RF
使用基于插头的微流体剂在纳米液液滴中检测细菌并确定其对抗生素的敏感性。
影响因子：
6.1
作者：
Boedicker JQ;Li L;Kline TR;Ismagilov RF
通讯作者：
Ismagilov RF