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Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

Felder E, Mossbrugger I, Lange M, Wölfel R - Toxins (Basel) (2012)

Bottom Line: 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.Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology.

View Article: PubMed Central - PubMed

Affiliation: Department for Medical Bio Reconnaissance and Verification, Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, Munich 80937, Germany. evafelder@bundeswehr.org

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. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

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Primers and Probes used in this study; the oligonucleotide binding sites within the genome of different lectin-producing plants are shown. The scale refers to the position on the genome of Ricinus communis (XM_002532143.1).
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toxins-04-00633-f002: Primers and Probes used in this study; the oligonucleotide binding sites within the genome of different lectin-producing plants are shown. The scale refers to the position on the genome of Ricinus communis (XM_002532143.1).

Mentions: The developed duplex qPCR assay for simultaneous detection of ricin and abrin DNA reliably amplified the targeted region from the genomic material of all cultivars of R. communis and A. precatorius. Results for these tests are summarized in Table 1. Ricin and abrin DNA could be distinguished by the differently labeled probes and the fluorescence signals for ricin (FAM) and abrin (Alx532) showed no crosstalk. Specific PCR products of 259 bp were confirmed by agarose gel electrophoresis in all tested positive samples (data not shown). DNA extracted from Abrus pulchellus, a plant producing the abrin-like toxin pulchellin, was also amplified and detected by the abrin-specific hybridization probe (see also the comparative alignment in Figure 2).


Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

Felder E, Mossbrugger I, Lange M, Wölfel R - Toxins (Basel) (2012)

Primers and Probes used in this study; the oligonucleotide binding sites within the genome of different lectin-producing plants are shown. The scale refers to the position on the genome of Ricinus communis (XM_002532143.1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475220&req=5

toxins-04-00633-f002: Primers and Probes used in this study; the oligonucleotide binding sites within the genome of different lectin-producing plants are shown. The scale refers to the position on the genome of Ricinus communis (XM_002532143.1).
Mentions: The developed duplex qPCR assay for simultaneous detection of ricin and abrin DNA reliably amplified the targeted region from the genomic material of all cultivars of R. communis and A. precatorius. Results for these tests are summarized in Table 1. Ricin and abrin DNA could be distinguished by the differently labeled probes and the fluorescence signals for ricin (FAM) and abrin (Alx532) showed no crosstalk. Specific PCR products of 259 bp were confirmed by agarose gel electrophoresis in all tested positive samples (data not shown). DNA extracted from Abrus pulchellus, a plant producing the abrin-like toxin pulchellin, was also amplified and detected by the abrin-specific hybridization probe (see also the comparative alignment in Figure 2).

Bottom Line: 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.Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology.

View Article: PubMed Central - PubMed

Affiliation: Department for Medical Bio Reconnaissance and Verification, Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, Munich 80937, Germany. evafelder@bundeswehr.org

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. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

Show MeSH