Limits...
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.

Show MeSH
Limiting dilution series of 107–100 copies DNA per reaction; NTC: no template control (a) A. precatorius DNA and (b) R. communis (cultivar Carmencita pink) DNA. For further use as positive control for verification of assay performance quantified PCR products were adjusted to result in final crossing point values of 30 and dried down in a SpeedVac vacuum centrifuge for long term storage.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3475220&req=5

toxins-04-00633-f003: Limiting dilution series of 107–100 copies DNA per reaction; NTC: no template control (a) A. precatorius DNA and (b) R. communis (cultivar Carmencita pink) DNA. For further use as positive control for verification of assay performance quantified PCR products were adjusted to result in final crossing point values of 30 and dried down in a SpeedVac vacuum centrifuge for long term storage.

Mentions: The 95% limit-of detection (LOD) as a common technical specification indicating the concentration down to which an assay will detect the analyte with at least 95% probability, was determined by amplification of quantified ricin and abrin DNA fragments (Figure 3). For ricin 10 copies DNA per reaction could be detected in 5 of 5 qPCR runs and 1 copy DNA per reaction could be detected in 1 of 5 qPCR runs. For abrin 10 copies DNA per reaction could be detected in 5 of 5 qPCR runs, 1 copy DNA per reaction could be detected in 4 of 5 qPCR runs and 0.5 copies DNA per reaction could be detected in 3 of 5 qPCR runs. The resulting 95% LOD was 3.0 genome copies per reaction for the detection of ricin and 1.2 genome copies per reaction for the detection of abrin (Figure 4).


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

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

Limiting dilution series of 107–100 copies DNA per reaction; NTC: no template control (a) A. precatorius DNA and (b) R. communis (cultivar Carmencita pink) DNA. For further use as positive control for verification of assay performance quantified PCR products were adjusted to result in final crossing point values of 30 and dried down in a SpeedVac vacuum centrifuge for long term storage.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

toxins-04-00633-f003: Limiting dilution series of 107–100 copies DNA per reaction; NTC: no template control (a) A. precatorius DNA and (b) R. communis (cultivar Carmencita pink) DNA. For further use as positive control for verification of assay performance quantified PCR products were adjusted to result in final crossing point values of 30 and dried down in a SpeedVac vacuum centrifuge for long term storage.
Mentions: The 95% limit-of detection (LOD) as a common technical specification indicating the concentration down to which an assay will detect the analyte with at least 95% probability, was determined by amplification of quantified ricin and abrin DNA fragments (Figure 3). For ricin 10 copies DNA per reaction could be detected in 5 of 5 qPCR runs and 1 copy DNA per reaction could be detected in 1 of 5 qPCR runs. For abrin 10 copies DNA per reaction could be detected in 5 of 5 qPCR runs, 1 copy DNA per reaction could be detected in 4 of 5 qPCR runs and 0.5 copies DNA per reaction could be detected in 3 of 5 qPCR runs. The resulting 95% LOD was 3.0 genome copies per reaction for the detection of ricin and 1.2 genome copies per reaction for the detection of abrin (Figure 4).

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