Guzmán Carolina, Jofre Juan, Blanch Anicet R, Lucena Francisco
Department of Microbiology, Biology Faculty, University of Barcelona, 645 Diagonal Avenue, 08028 Barcelona, Spain.
J Virol Methods. 2007 Sep;144(1-2):41-8. doi: 10.1016/j.jviromet.2007.03.017. Epub 2007 May 11.
Extraction of viruses and bacteriophages from sludge, soil and treated biowaste requires homogenization, elution, clarification and detoxification-decontamination steps. Seeding these matrixes with bacteriophages does not reproduce what happens in nature. Therefore, naturally occurring matrixes, raw sludge, digested and dewatered sludge and compost, containing high numbers of somatic coliphages, and soils contaminated with wastewater or raw sludge were used in the extraction assays. Based on eluting the bacteriophages with beef extract, a feasible method in which the different steps had been optimized has been established. The method is feasible, repeatable, robust and applicable in routine laboratories. Digested and dewatered sludge has been probed to be useful as a reference material for validation studies and for "in lab" quality control. The established method includes homogenization by magnetic stirring, elution (which is performed at the same time that homogenization) with 10% beef extract at neutral pH, clarification by centrifuging at 4000 x g and decontamination by filtration through low protein binding 0.22 microm diameter pore size membrane filters.
从污泥、土壤和处理后的生物废弃物中提取病毒和噬菌体需要经过匀浆、洗脱、澄清以及解毒-去污等步骤。在这些基质中接种噬菌体并不能重现自然发生的情况。因此，提取试验使用了含有大量体细胞噬菌体的天然基质、生污泥、消化和脱水污泥以及堆肥，以及被废水或生污泥污染的土壤。基于用牛肉膏洗脱噬菌体，建立了一种对不同步骤进行了优化的可行方法。该方法可行、可重复、稳健且适用于常规实验室。已探测到消化和脱水污泥可作为验证研究和“实验室内部”质量控制的参考材料。所建立的方法包括通过磁力搅拌进行匀浆，在中性pH下用10%牛肉膏进行洗脱（与匀浆同时进行），以4000×g离心进行澄清，以及通过低蛋白结合的0.22微米孔径膜过滤器过滤进行去污。
