Rapid Filtration Separation-Based Sample Preparation Method for Bacillus Spores in Powdery and Environmental Matrices

Abstract

Authorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA. Bacillus atrophaeus subsp. globigii was used as a simulant of Bacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed the dual-filter method for applied recovery of microbial particles from environmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of <10 min. This sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.

Selective isolation of bacteria for metagenomic analysis: Impact of membrane characteristics on bacterial filterability

“…68 Filters with pore diameters between 5 and 10 lm are suitable for the collection of bacteria in the filtrate as they are able to allow average-size bacteria to pass through to the filtrate while retaining larger particles. 113 Batch consistency and even choice of pore size from a given manufacturer should be considered in membrane filtration. This is because the chemical and physical composition of membrane filters from each manufacturer might differ and affect membrane filtration.…”

Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on “Road Closure”

“…To our knowledge, no lateral-flow based techniques for spore detection that directly analyze such high levels of powdery samples without any pre-treatment have been reported. Although PCR provides similar levels of sensitivity when testing 2% starch samples and milk samples, it fails to detect B. anthracis spores in baking soda solution at this concentration (Isabel et al, 2012). These tests thus demonstrate that our lateral-flow immunoassay is not affected by interference and performs excellently even when there is a high content of powdery matrix.…”

Rapid and Efficient Filtration-Based Procedure for Separation and Safe Analysis of CBRN Mixed Samples

“…The TAT required to achieve a complete separation process of CB mixed samples within simple and complex matrices was less than 10 minutes, which is alike the TAT required to carry out a dual filtration process for recovering bacterial spores from environmental and powdery samples [19]. At last and as demonstrated here both with liquid and complex matrices, the current method is not only able to recover spores and vegetative bacteria but also nanosized biological particles like bacteriophages MS2.…”

“…Two previous reports described the use of filtration as a method for recovering spores of Bacillus from suspicious powdery and environmental matrices or for quantifying and detecting Salmonella in biological samples [19], [20]. However, none of these reports addressed the issue of separating CB mixed samples nor did they assess nanosized biological agents.…”