title: Rapid and efficient filtration-based procedure for separation and safe analysis of CBRN mixed samples.
abstract: Separating CBRN mixed samples that contain both chemical and biological warfare agents (CB mixed sample) in liquid and solid matrices remains a very challenging issue. Parameters were set up to assess the performance of a simple filtration-based method first optimized on separate C- and B-agents, and then assessed on a model of CB mixed sample. In this model, MS2 bacteriophage, Autographa californica nuclear polyhedrosis baculovirus (AcNPV), Bacillus atrophaeus and Bacillus subtilis spores were used as biological agent simulants whereas ethyl methylphosphonic acid (EMPA) and pinacolyl methylphophonic acid (PMPA) were used as VX and soman (GD) nerve agent surrogates, respectively. Nanoseparation centrifugal devices with various pore size cut-off (30 kD up to 0.45 µm) and three RNA extraction methods (Invisorb, EZ1 and Nuclisens) were compared. RNA (MS2) and DNA (AcNPV) quantification was carried out by means of specific and sensitive quantitative real-time PCRs (qPCR). Liquid chromatography coupled to time-of-flight mass spectrometry (LC/TOFMS) methods was used for quantifying EMPA and PMPA. Culture methods and qPCR demonstrated that membranes with a 30 kD cut-off retain more than 99.99% of biological agents (MS2, AcNPV, Bacillus Atrophaeus and Bacillus subtilis spores) tested separately. A rapid and reliable separation of CB mixed sample models (MS2/PEG-400 and MS2/EMPA/PMPA) contained in simple liquid or complex matrices such as sand and soil was also successfully achieved on a 30 kD filter with more than 99.99% retention of MS2 on the filter membrane, and up to 99% of PEG-400, EMPA and PMPA recovery in the filtrate. The whole separation process turnaround-time (TAT) was less than 10 minutes. The filtration method appears to be rapid, versatile and extremely efficient. The separation method developed in this work constitutes therefore a useful model for further evaluating and comparing additional separation alternative procedures for a safe handling and preparation of CB mixed samples.
authors: Biothreats Unit, Defense Laboratories Department (DLD), Belgian Armed Forces, Brussels, Belgium.
authors: Mostafa
authors: M
authors: Bentahir
authors: Chemical Analysis Laboratory, Defense Laboratories Department (DLD), Belgian Armed Forces, Brussels, Belgium.
authors: Frederic
authors: F
authors: Laduron
authors: Biothreats Unit, Defense Laboratories Department (DLD), Belgian Armed Forces, Brussels, Belgium.
authors: Leonid
authors: L
authors: Irenge
authors: Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium.
authors: Jérôme
authors: J
authors: Ambroise
authors: Biothreats Unit, Defense Laboratories Department (DLD), Belgian Armed Forces, Brussels, Belgium ; Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium.
authors: Jean-Luc
authors: JL
authors: Gala
mesh_terms: D001407:Bacillus / Q000302:isolation & purification*; D016367:Baculoviridae / Q000302:isolation & purification*; D002619:Chemical Warfare Agents / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D005374:Filtration / Q000295:instrumentation*; D017909:Levivirus / Q000302:isolation & purification*; D013058:Mass Spectrometry; D063065:Organophosphonates / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D012988:Soil Microbiology; D012999:Soman / Q000302:isolation & purification; D013171:Spores, Bacterial / Q000302:isolation & purification*
chemical_list: D002619:Chemical Warfare Agents; D063065:Organophosphonates; C108318:ethyl methylphosphonic acid; D012999:Soman