Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
<p>Abstract</p> <p>Background</p> <p>The limited circulation of many of the agents that are likely to be used in a bioterrorism attack precludes the ready availability of positive controls. This means that only specialized laboratories can screen for the presence of the...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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BMC
2004-05-01
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| Series: | BMC Microbiology |
| Online Access: | http://www.biomedcentral.com/1471-2180/4/21 |
| _version_ | 1818807605978464256 |
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| author | La Scola Bernard Charrel Remy N Raoult Didier |
| author_facet | La Scola Bernard Charrel Remy N Raoult Didier |
| author_sort | La Scola Bernard |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>The limited circulation of many of the agents that are likely to be used in a bioterrorism attack precludes the ready availability of positive controls. This means that only specialized laboratories can screen for the presence of these agents by nucleic amplification assays. Calibrated controls are also necessary for quantitative measurements. Primers and probes to be used in both conventional and real-time PCR assays were designed for the detection of agents likely to be used by a bioterrorist. Three plasmids, each of which contains 4 to 6 specific sequences from agents on the CDC Category A and B list (excluding RNA viruses) were constructed. Two plasmids incorporate the sequences of Category A and B agents, respectively. The third plasmid incorporates sequences from Variola major and organisms that cause rash-like illnesses that may be clinically confused with smallpox. An "exogenic sequence", introducing a <it>Not</it>I restriction site was incorporated in the native sequences of the bioterrorism agents inserted in plasmids. The designed molecular system for detection of bioterrorism agents was tested on each of these agents (except Monkeypox virus, Smallpox virus and 2 <it>Burkholderia </it>species for which no native DNA was available) and a collection of 50 isolates of <it>C. burnetii </it>using constructed plasmids as positive controls.</p> <p>Results</p> <p>Designed primers and probes allowed molecular detection, in either single or multiplex assays, of agent-specific targets with analytical sensitivities of between 1 and 100 DNA copies. The plasmids could be used as positive controls. False-positive results due to contamination by the positive control were easily detected by sequencing and eliminated by digestion with <it>Not</it>I.</p> <p>Conclusion</p> <p>Plasmid A and B can be used as positive controls in molecular assays for the detection of bioterrorism agents in clinical specimens or environmental samples. Plasmid C can be used as a positive control in differentiation of vesicular rashes. It is also possible to avoid or to ensure immediate detection of false positive results due to contamination by positive controls using these plasmids. These plasmids and the corresponding primers and probes are immediately available for all clinical microbiology laboratories provided they have molecular amplification equipment.</p> |
| first_indexed | 2024-12-18T19:28:20Z |
| format | Article |
| id | doaj.art-c2b5649394224a02a768748348fe7e44 |
| institution | Directory Open Access Journal |
| issn | 1471-2180 |
| language | English |
| last_indexed | 2024-12-18T19:28:20Z |
| publishDate | 2004-05-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Microbiology |
| spelling | doaj.art-c2b5649394224a02a768748348fe7e442022-12-21T20:55:48ZengBMCBMC Microbiology1471-21802004-05-01412110.1186/1471-2180-4-21Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorismLa Scola BernardCharrel Remy NRaoult Didier<p>Abstract</p> <p>Background</p> <p>The limited circulation of many of the agents that are likely to be used in a bioterrorism attack precludes the ready availability of positive controls. This means that only specialized laboratories can screen for the presence of these agents by nucleic amplification assays. Calibrated controls are also necessary for quantitative measurements. Primers and probes to be used in both conventional and real-time PCR assays were designed for the detection of agents likely to be used by a bioterrorist. Three plasmids, each of which contains 4 to 6 specific sequences from agents on the CDC Category A and B list (excluding RNA viruses) were constructed. Two plasmids incorporate the sequences of Category A and B agents, respectively. The third plasmid incorporates sequences from Variola major and organisms that cause rash-like illnesses that may be clinically confused with smallpox. An "exogenic sequence", introducing a <it>Not</it>I restriction site was incorporated in the native sequences of the bioterrorism agents inserted in plasmids. The designed molecular system for detection of bioterrorism agents was tested on each of these agents (except Monkeypox virus, Smallpox virus and 2 <it>Burkholderia </it>species for which no native DNA was available) and a collection of 50 isolates of <it>C. burnetii </it>using constructed plasmids as positive controls.</p> <p>Results</p> <p>Designed primers and probes allowed molecular detection, in either single or multiplex assays, of agent-specific targets with analytical sensitivities of between 1 and 100 DNA copies. The plasmids could be used as positive controls. False-positive results due to contamination by the positive control were easily detected by sequencing and eliminated by digestion with <it>Not</it>I.</p> <p>Conclusion</p> <p>Plasmid A and B can be used as positive controls in molecular assays for the detection of bioterrorism agents in clinical specimens or environmental samples. Plasmid C can be used as a positive control in differentiation of vesicular rashes. It is also possible to avoid or to ensure immediate detection of false positive results due to contamination by positive controls using these plasmids. These plasmids and the corresponding primers and probes are immediately available for all clinical microbiology laboratories provided they have molecular amplification equipment.</p>http://www.biomedcentral.com/1471-2180/4/21 |
| spellingShingle | La Scola Bernard Charrel Remy N Raoult Didier Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism BMC Microbiology |
| title | Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism |
| title_full | Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism |
| title_fullStr | Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism |
| title_full_unstemmed | Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism |
| title_short | Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism |
| title_sort | multi pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism |
| url | http://www.biomedcentral.com/1471-2180/4/21 |
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