Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Posit...

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Bibliographic Details
Published in:	Journal of applied microbiology Vol. 115; no. 1; pp. 156 - 162
Main Authors:	Thomas, M.C., Shields, M.J., Hahn, K.R., Janzen, T.W., Goji, N., Amoako, K.K.
Format:	Journal Article
Language:	English
Published:	Oxford Blackwell 01-07-2013 Oxford University Press
Subjects:	Bacillus anthracis Bacillus anthracis - genetics Biological and medical sciences DNA extraction DNA, Bacterial - analysis DNA, Bacterial - isolation & purification food Food Microbiology Fundamental and applied biological sciences. Psychology Immunomagnetic Separation Malus Microbiology Real-Time Polymerase Chain Reaction spores Spores, Bacterial - genetics Bacillus anthracis Evaluation Immunomagnetic method DNA extraction Applied microbiology Sample immunomagnetic separation Foodstuff Bacillaceae Spores Bacillales DNA Bacteria Isolate Extraction Food
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Summary:	Aims Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 101 and 1·3 × 102 CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS). Methods and Results The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors. Conclusions Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit. Significance and Impact of the Study The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1364-5072 1365-2672
DOI:	10.1111/jam.12206