Limits...
Comparison of two suspension arrays for simultaneous detection of five biothreat bacterial in powder samples.

Yang Y, Wang J, Wen H, Liu H - J. Biomed. Biotechnol. (2012)

Bottom Line: Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms.Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria.The two suspension arrays can be utilized to detect Bacillus anthracis sterne spore and Yersinia pestis EV76 from mimic "write powder" samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100123, China.

ABSTRACT
We have developed novel Bio-Plex assays for simultaneous detection of Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis, and Burkholderia pseudomallei. Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms. The other assay is based on multiplex PCR to simultaneously amplify five species-specific pathogen identification-targeted regions unique to individual pathogen. Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria. However, universal primer PCR-based array could not identify Bacillus anthracis, Yersinia pestis, and Brucella spp. at the species level because of the high conservation of 16S rDNA of the same genus. The two suspension arrays can be utilized to detect Bacillus anthracis sterne spore and Yersinia pestis EV76 from mimic "write powder" samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

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Comparison of detection sensitivity of multiplex PCR-based and universal primers PCR-coupled assay, the inlet shows the cut-off value of two assays. (a) Bacillus anthracis; (b) Yersinia pestis; (c) Francisella tularensis; (d) Brucella spp.; (e) Burkholderia pseudomallei.
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Related In: Results  -  Collection


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fig3: Comparison of detection sensitivity of multiplex PCR-based and universal primers PCR-coupled assay, the inlet shows the cut-off value of two assays. (a) Bacillus anthracis; (b) Yersinia pestis; (c) Francisella tularensis; (d) Brucella spp.; (e) Burkholderia pseudomallei.

Mentions: The limits of detection for each bacterium were tested in universal primer PCR-based array and multiplex PCR-based array. Figure 3 shows the limits of detection for each set of primers when tested universal primer PCR and multiplex PCR in individual species. We observed that a semilogarithm dose-response curve between the MFI and DNA concentration followed a dynamic range. The universal primer PCR-coupled liquid bead array was capable of detecting the specific target sequence when a minimum amount of 0.8 pg Burkholderia pseudomallei, 40 pg Brucella spp., 14 pg Bacillus anthracis, 0.2 pg Francisella tularensis, or 2.2 pg Yersinia pestis genomic DNA template was present in the PCR amplification reactions; the multiplex PCR-suspension array was sensitive with a detection limit of 0.62 pg Burkholderia pseudomallei, 22.5 pg Brucella spp., 70 pg Bacillus anthracis, 0.95 pg Francisella tularensis, and 50 fg Yersinia pestis genomic DNA template. A negative control was added as previously described [9].


Comparison of two suspension arrays for simultaneous detection of five biothreat bacterial in powder samples.

Yang Y, Wang J, Wen H, Liu H - J. Biomed. Biotechnol. (2012)

Comparison of detection sensitivity of multiplex PCR-based and universal primers PCR-coupled assay, the inlet shows the cut-off value of two assays. (a) Bacillus anthracis; (b) Yersinia pestis; (c) Francisella tularensis; (d) Brucella spp.; (e) Burkholderia pseudomallei.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368695&req=5

fig3: Comparison of detection sensitivity of multiplex PCR-based and universal primers PCR-coupled assay, the inlet shows the cut-off value of two assays. (a) Bacillus anthracis; (b) Yersinia pestis; (c) Francisella tularensis; (d) Brucella spp.; (e) Burkholderia pseudomallei.
Mentions: The limits of detection for each bacterium were tested in universal primer PCR-based array and multiplex PCR-based array. Figure 3 shows the limits of detection for each set of primers when tested universal primer PCR and multiplex PCR in individual species. We observed that a semilogarithm dose-response curve between the MFI and DNA concentration followed a dynamic range. The universal primer PCR-coupled liquid bead array was capable of detecting the specific target sequence when a minimum amount of 0.8 pg Burkholderia pseudomallei, 40 pg Brucella spp., 14 pg Bacillus anthracis, 0.2 pg Francisella tularensis, or 2.2 pg Yersinia pestis genomic DNA template was present in the PCR amplification reactions; the multiplex PCR-suspension array was sensitive with a detection limit of 0.62 pg Burkholderia pseudomallei, 22.5 pg Brucella spp., 70 pg Bacillus anthracis, 0.95 pg Francisella tularensis, and 50 fg Yersinia pestis genomic DNA template. A negative control was added as previously described [9].

Bottom Line: Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms.Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria.The two suspension arrays can be utilized to detect Bacillus anthracis sterne spore and Yersinia pestis EV76 from mimic "write powder" samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100123, China.

ABSTRACT
We have developed novel Bio-Plex assays for simultaneous detection of Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis, and Burkholderia pseudomallei. Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms. The other assay is based on multiplex PCR to simultaneously amplify five species-specific pathogen identification-targeted regions unique to individual pathogen. Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria. However, universal primer PCR-based array could not identify Bacillus anthracis, Yersinia pestis, and Brucella spp. at the species level because of the high conservation of 16S rDNA of the same genus. The two suspension arrays can be utilized to detect Bacillus anthracis sterne spore and Yersinia pestis EV76 from mimic "write powder" samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

Show MeSH
Related in: MedlinePlus