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Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism.

Charrel RN, La Scola B, Raoult D - BMC Microbiol. (2004)

Bottom Line: False-positive results due to contamination by the positive control were easily detected by sequencing and eliminated by digestion with NotI.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité des Rickettsies, CNRS UMR 6020 IFR 48, Faculté de Médecine, Marseille, France. rnc-virophdm@pop.gulliver.fr

ABSTRACT

Background: 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 NotI 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 Burkholderia species for which no native DNA was available) and a collection of 50 isolates of C. burnetii using constructed plasmids as positive controls.

Results: 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 NotI.

Conclusion: 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.

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Molecular detection and identification of true and false positives using plasmid CatA as positive control. 3a. NotI predigestion of CatA plasmid DNA. A, B, C, D correspond to Not I digested-CatA plasmid DNA (overnight digestion with 10 U followed by an 1-hour digestion with another 10 U) respectively PCR amplified with Y. pestis, F. tularensis, B. anthracis, and smallpox virus oligonucleotide primers. E, F, G, H correspond to the same DNA samples which have not been digested by NotI. 3b. Discrimination of Y. pestis amplicons by NotI post-PCR digestion in a 3% agarose gel. lane I, NotI post-PCR digestion of amplicon from Y. pestis native DNA (101 bp); lane J, amplicon from CatA plasmid DNA (113 bp); lane K, NotI post-PCR digestion of amplicon from CatA plasmid DNA (digestion result in 2 products of 76 bp and 37 bp, respectively; only the 76 bp product is seen). 3c. Melting curves obtained from SYBR Green Light Cycler reactions. Curve A was obtained with CatA plasmid DNA and Y. pestis primers; curve B was obtained with Y. pestis DNA amplified with Y. pestis specific primers; curve C was obtained with Y. pestis DNA amplified in a multiplex reaction including the 8 primers specific for the category A agents (Y. pestis, F. tularensis, B. anthracis, smallpox virus). The melting temperature observed with A is 82.65 whereas it is 81.97 with native Y. pestis DNA (curves B and C).
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Figure 3: Molecular detection and identification of true and false positives using plasmid CatA as positive control. 3a. NotI predigestion of CatA plasmid DNA. A, B, C, D correspond to Not I digested-CatA plasmid DNA (overnight digestion with 10 U followed by an 1-hour digestion with another 10 U) respectively PCR amplified with Y. pestis, F. tularensis, B. anthracis, and smallpox virus oligonucleotide primers. E, F, G, H correspond to the same DNA samples which have not been digested by NotI. 3b. Discrimination of Y. pestis amplicons by NotI post-PCR digestion in a 3% agarose gel. lane I, NotI post-PCR digestion of amplicon from Y. pestis native DNA (101 bp); lane J, amplicon from CatA plasmid DNA (113 bp); lane K, NotI post-PCR digestion of amplicon from CatA plasmid DNA (digestion result in 2 products of 76 bp and 37 bp, respectively; only the 76 bp product is seen). 3c. Melting curves obtained from SYBR Green Light Cycler reactions. Curve A was obtained with CatA plasmid DNA and Y. pestis primers; curve B was obtained with Y. pestis DNA amplified with Y. pestis specific primers; curve C was obtained with Y. pestis DNA amplified in a multiplex reaction including the 8 primers specific for the category A agents (Y. pestis, F. tularensis, B. anthracis, smallpox virus). The melting temperature observed with A is 82.65 whereas it is 81.97 with native Y. pestis DNA (curves B and C).

Mentions: Thirteen viral and bacterial targeted agents were detected by using the primers and probes developed in this study using standard PCR, Taq Man and Light Cycler real-time PCRs (Table 1 and 2). Four agents were not available (Monkeypox, Smallpox virus and 2 Burkholderia species). Only the specific primers with the corresponding DNA gave positive results. Multiplex real-time PCR results were identical. The 15 targeted plasmid sequences were detected using the same primers. Amplicons were differentiated from the native DNA by aspect of melting curves and sequencing of the amplifiates (Figure 3, additional file 2). All 50 C. burnetii strains were detected using the same procedures.


Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism.

Charrel RN, La Scola B, Raoult D - BMC Microbiol. (2004)

Molecular detection and identification of true and false positives using plasmid CatA as positive control. 3a. NotI predigestion of CatA plasmid DNA. A, B, C, D correspond to Not I digested-CatA plasmid DNA (overnight digestion with 10 U followed by an 1-hour digestion with another 10 U) respectively PCR amplified with Y. pestis, F. tularensis, B. anthracis, and smallpox virus oligonucleotide primers. E, F, G, H correspond to the same DNA samples which have not been digested by NotI. 3b. Discrimination of Y. pestis amplicons by NotI post-PCR digestion in a 3% agarose gel. lane I, NotI post-PCR digestion of amplicon from Y. pestis native DNA (101 bp); lane J, amplicon from CatA plasmid DNA (113 bp); lane K, NotI post-PCR digestion of amplicon from CatA plasmid DNA (digestion result in 2 products of 76 bp and 37 bp, respectively; only the 76 bp product is seen). 3c. Melting curves obtained from SYBR Green Light Cycler reactions. Curve A was obtained with CatA plasmid DNA and Y. pestis primers; curve B was obtained with Y. pestis DNA amplified with Y. pestis specific primers; curve C was obtained with Y. pestis DNA amplified in a multiplex reaction including the 8 primers specific for the category A agents (Y. pestis, F. tularensis, B. anthracis, smallpox virus). The melting temperature observed with A is 82.65 whereas it is 81.97 with native Y. pestis DNA (curves B and C).
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Figure 3: Molecular detection and identification of true and false positives using plasmid CatA as positive control. 3a. NotI predigestion of CatA plasmid DNA. A, B, C, D correspond to Not I digested-CatA plasmid DNA (overnight digestion with 10 U followed by an 1-hour digestion with another 10 U) respectively PCR amplified with Y. pestis, F. tularensis, B. anthracis, and smallpox virus oligonucleotide primers. E, F, G, H correspond to the same DNA samples which have not been digested by NotI. 3b. Discrimination of Y. pestis amplicons by NotI post-PCR digestion in a 3% agarose gel. lane I, NotI post-PCR digestion of amplicon from Y. pestis native DNA (101 bp); lane J, amplicon from CatA plasmid DNA (113 bp); lane K, NotI post-PCR digestion of amplicon from CatA plasmid DNA (digestion result in 2 products of 76 bp and 37 bp, respectively; only the 76 bp product is seen). 3c. Melting curves obtained from SYBR Green Light Cycler reactions. Curve A was obtained with CatA plasmid DNA and Y. pestis primers; curve B was obtained with Y. pestis DNA amplified with Y. pestis specific primers; curve C was obtained with Y. pestis DNA amplified in a multiplex reaction including the 8 primers specific for the category A agents (Y. pestis, F. tularensis, B. anthracis, smallpox virus). The melting temperature observed with A is 82.65 whereas it is 81.97 with native Y. pestis DNA (curves B and C).
Mentions: Thirteen viral and bacterial targeted agents were detected by using the primers and probes developed in this study using standard PCR, Taq Man and Light Cycler real-time PCRs (Table 1 and 2). Four agents were not available (Monkeypox, Smallpox virus and 2 Burkholderia species). Only the specific primers with the corresponding DNA gave positive results. Multiplex real-time PCR results were identical. The 15 targeted plasmid sequences were detected using the same primers. Amplicons were differentiated from the native DNA by aspect of melting curves and sequencing of the amplifiates (Figure 3, additional file 2). All 50 C. burnetii strains were detected using the same procedures.

Bottom Line: False-positive results due to contamination by the positive control were easily detected by sequencing and eliminated by digestion with NotI.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité des Rickettsies, CNRS UMR 6020 IFR 48, Faculté de Médecine, Marseille, France. rnc-virophdm@pop.gulliver.fr

ABSTRACT

Background: 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 NotI 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 Burkholderia species for which no native DNA was available) and a collection of 50 isolates of C. burnetii using constructed plasmids as positive controls.

Results: 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 NotI.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus