title: Evaluation of DNA extraction methods for Bacillus anthracis spores spiked to food and feed matrices at biosafety level 3 conditions.
abstract: The DNA extraction efficiency from milk, whey, soy, corn gluten meal, wheat powders and heat-treated corn grain that were spiked with Bacillus anthracis and Bacillus thuringiensis spores was determined. Two steps were critical: lysis of the spores and binding of the free DNA to the DNA binding magnetic beads in the presence of the interfering powders. For the guanidine-thiocyanate based Nuclisens lysis buffer from Biomerieux we found that between 15 and 30% of the spores survived the lysis step. As most lysis buffers in DNA/RNA extraction kits are guanidine based it is likely that other lysis buffers will show a similar partial lysis of the Bacillus spores. Our results show that soybean flour and wheat flour inhibited the DNA extraction process strongest, leading to unreliable DNA extractions when using too much of the matrix. For corn gluten meal, heat-treated corn grain and milk powders, DNA extraction efficiencies in the presence of 100mg and 10mg of powder resulted in 70%-95% reduced DNA recoveries. The inhibition was, however, reliable and intermediate compared to the inhibition by soy and wheat. Whey powder had the lowest inhibitory effect on DNA-extraction efficiency and recoveries of 70-100% could be reached when using 10mg of powder. The results show that reducing the amount of matrix leads to better DNA-extraction efficiencies, particularly for strongly inhibiting powders such as soy and wheat. Based on these results, a standard protocol to directly isolate DNA from micro-organisms present in complex matrixes such as food and feed powders was designed.
authors.affiliation: National Institute for Public Health and the Environment (RIVM), Centre for Infectious Disease Control (CIb), Laboratory for Zoonoses and Environmental Microbiology (LZO), Antonie van Leeuwenhoeklaan 9, P.O. Box 1, Bilthoven, The Netherlands. peter.wielinga@rivm.nl
authors.forename: Peter R
authors.initials: PR
authors.lastname: Wielinga
authors.forename: Lianne
authors.initials: L
authors.lastname: de Heer
authors.forename: Astrid
authors.initials: A
authors.lastname: de Groot
authors.forename: Raditijo A
authors.initials: RA
authors.lastname: Hamidjaja
authors.forename: Geert
authors.initials: G
authors.lastname: Bruggeman
authors.forename: Kieran
authors.initials: K
authors.lastname: Jordan
authors.forename: Bart J
authors.initials: BJ
authors.lastname: van Rotterdam
mesh_terms: D000818:Animals; D001408:Bacillus anthracis / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D001413:Bacillus thuringiensis / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D015373:Bacterial Typing Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D005433:Flour / Q000382:microbiology; D005516:Food Microbiology; D008892:Milk / Q000382:microbiology; D017422:Sequence Analysis, DNA; D045730:Soy Foods / Q000382:microbiology; D013171:Spores, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014908:Triticum / Q000382:microbiology; D003313:Zea mays / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial