A bacteriophage-based platform for rapid trace detection of proteases.

Sensitive, inexpensive and rapid protease activity assays are of great merit for clinical diagnostics. Detection of protease-based toxins produced by Clostridium botulinum and Bacillus anthracis represents a particularly challenging task as exceptional sensitivity is a prerequisite due to the extreme potency of the toxins. Here, we present an inexpensive and sensitive assay platform for activity-based protease quantification utilizing filamentous bacteriophage as an exponentially amplifiable reporter and its application to detection of these bacterial toxins. The assay is based on specific cleavage of bacteriophage from a solid support and its subsequent quantification by means of infectivity or quantitative PCR. Detection of botulinum neurotoxin (BoNT) serotypes A and B and anthrax lethal factor in the pM range was demonstrated with a limit of detection of BoNT/A under optimized conditions of 2 pM.

灵敏、廉价且快速的蛋白酶活性检测方法在临床诊断中具有重要价值。肉毒梭菌和炭疽芽孢杆菌所产生的基于蛋白酶的毒素的检测是一项极具挑战性的任务，因为这些毒素毒性极强，所以极高的灵敏度是一个先决条件。在此，我们提出了一种廉价且灵敏的基于活性的蛋白酶定量检测平台，该平台利用丝状噬菌体作为可指数扩增的报告分子，并将其应用于这些细菌毒素的检测。该检测方法基于噬菌体从固体支持物上的特异性裂解，然后通过感染性或定量PCR对其进行定量。我们证明了该方法能够检测皮摩尔（pM）范围内的A型和B型肉毒神经毒素（BoNT）以及炭疽致死因子，在优化条件下，BoNT/A的检测限为2皮摩尔。