Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR) 英文参考文献.docVIP

Toxins 2012, 4, 633-642; doi:10.3390/toxins4090633 OPEN ACCESS toxins ISSN 2072-6651 /journal/toxins Article Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR) Eva Felder, Ilona Mossbrugger, Mirko Lange and Roman W?lfel * Department for Medical Bio Reconnaissance and Verification, Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, Munich 80937, Germany; E-Mails: evafelder@ (E.F.); ilonamossbrugger@ (I.M.); mirko1lange@ (M.L.) * Author to whom correspondence should be addressed; E-Mail: romanwoelfel@; Tel.: +49-89-3168-3894; Fax: +49-89-3168-3983. Received: 14 July 2012; in revised form: 14 August 2012 / Accepted: 16 August 2012 / Published: 31 August 2012 Abstract: Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. Th