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Title: Application of a real time PCR method to detect castor toxin contamination in fluid milk and eggs

Author
item He, Xiaohua
item Carter, John
item Brandon, David
item Cheng, Luisa
item McKeon, Thomas

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/6/2007
Publication Date: 7/25/2007
Citation: He, X., Carter, J.M., Brandon, D.L., Cheng, L.W., Mckeon, T.A. 2007. Application of a real time PCR method to detect castor toxin contamination in fluid milk and eggs. Journal of Agricultural and Food Chemistry. 55:6897-6902

Interpretive Summary: The intentional adulteration of food with ricin as an instrument of bioterrorism is a matter of increasing concern. Because crude castor seed preparations could be used as a bioweapon, we developed a screening strategy that employs sensitive detection of the DNA of the castor plant, rather than the protein toxin itself. This assay could be formatted as part of a kit to detect multiple biothreats simultaneously.

Technical Abstract: The castor seed contains ricin, which is one of the most potent biological toxins and is widely considered to be a threat agent for bioterrorism. In this study, a rapid and sensitive PCR method was developed for the detection of castor contamination in milk and liquid egg samples. Primers targeting ricin gene sequences not found in either the bovine or chicken genome were employed, and primers against a highly conserved sequence from the 18S ribosomal RNA gene were used as a positive control for DNA extraction and PCR reaction efficiency. The quantity and quality of DNA prepared from castor spiked or non-spiked milk and egg samples obtained from three different DNA extraction methods were compared. The cetyl trimethylammonium bromide (CTAB) method yielded the highest quality of DNA and is most suitable for the sensitive detection of castor DNA by real-time PCR in both milk and liquid egg matrices. However, taking time and cost into consideration, a commercial kit designed for extraction of DNA from stool could be used as an alternative method for the routine extraction of DNA from milk for real-time PCR assays. The egg matrix was found to inhibit PCR amplification and interfere with two of the three methods tested for DNA extraction. Egg yolk had a larger negative effect on PCR amplification than the egg white matrix. Both real-time PCR systems developed in this study, TaqMan and SYBR Green I dye, were capable of detecting <0.00001% of castor acetone powder, corresponding to 5 ng of ricin in 1 mL milk or liquid egg, well below the toxic dose for humans. The real-time PCR method could be used as a fast and accurate alternate detection method for intentional castor contamination of food matrices.