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.

Fig 1

DARE procedure and DNA extraction. The complete procedure required 10 steps, 10 tools, and 10 min. Materials are identified as follows: A, swab; F, filter; S, syringe; T, tube. The DARE procedure is executed in the first eight steps. Steps 9 and 10 represent the standardized technique with the BD GeneOhm lysis kit, which includes glass bead-beating lysis and a heating step. Red indicates that tubes or syringes contain bacteria.

Fig 2

Penetration and expulsion of bacterial spores in 0.45-μm-pore-size filters. We studied the penetration and expulsion of FITC-labeled B. atrophaeus spores within the 0.45-μm-pore-size Millex-HV filters by using confocal microscopy. The DARE procedure excluding (A) or including (B) the bacterial recovery step (Fig. 1, step 8) was executed. The filters were observed from the side facing the tube, and the positions of spores were measured from the surfaces of the filters. The numbers of spores in five FOVs for each 4-μm-deep stratum were calculated and are shown per surface unit. Asterisks indicate a <5% probability of finding a bacterium at a particular depth interval.