Evaluation of DNA extraction methods for Bacillus anthracis spores spiked to food and feed matrices at biosafety level 3 conditions

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 magne...

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Bibliographic Details
Published in:International journal of food microbiology 2011-11, Vol.150 (2), p.122-127
Main Authors: Wielinga, Peter R., de Heer, Lianne, de Groot, Astrid, Hamidjaja, Raditijo A., Bruggeman, Geert, Jordan, Kieran, van Rotterdam, Bart J.
Format: Article
Language:English
Subjects:
Animals
Anthrax
Bacillus anthracis
Bacillus anthracis - genetics
Bacillus anthracis - growth & development
Bacillus anthracis - isolation & purification
Bacillus thuringiensis
Bacillus thuringiensis - genetics
Bacillus thuringiensis - growth & development
Bacillus thuringiensis - isolation & purification
Bacterial Typing Techniques - methods
DNA
DNA extraction efficiency
DNA, Bacterial - analysis
DNA, Bacterial - chemistry
DNA, Bacterial - genetics
Flour - microbiology
Food
Food Microbiology
Gluten
Grain
Guanidine
Milk
Milk - microbiology
PCR inhibitors
Powder
Quantitative multiplex real-time PCR
RNA
Sequence Analysis, DNA
Soy Foods - microbiology
Soybeans
Spores
Spores, Bacterial - chemistry
Spores, Bacterial - genetics
Spores, Bacterial - isolation & purification
Triticum - microbiology
Triticum aestivum
Whey
Zea mays - microbiology
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Summary: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 100 mg and 10 mg 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 10 mg 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. ► DNA extraction and PCR efficiency for B. anthracis spores spiked to 10 food and feed powders. ► In particular DNA extraction and not PCR efficiency was affected by the powders. ► For strongly interfering powders only reduction of the amount of powder made DNA extraction possible. ► When unfamiliar with the matrix, use a minimal amount of powder e.g. 10 mg.
ISSN:0168-1605
1879-3460
DOI:10.1016/j.ijfoodmicro.2011.07.023