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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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Specificity test of probes in multiplex primer PCR-coupled suspension array. For each tested agent (x-axis), the response of each of six beads is shown (y-axis). Response is given as the median fluorescent intensity (MFI) at z-axis. Dotted bars indicate the probe of BA-1-coated bead; grid bars correspond to BA-2; right-slash bars are Yp-2; straight bars are Bru-2; right-dot bars indicate Ft-2; zigzag bars are Bp-2. From the y-axis, B is the blank blocks; Ba is Bacillus anthracis Sterne; Yp is Yersinia pestis EV76; Bru is Brucella spp. M5; Ft is Francisella tularensis and Bp is Burkholderia pseudomallei. The six bacterial coated beads are from species Bacillus anthracis, Yersinia pestis, Brucella abortus, Francisella tularensis, and Burkholderia pseudomallei, respectively. Each sample yields an appropriate response from each of the six beads present.
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fig4: Specificity test of probes in multiplex primer PCR-coupled suspension array. For each tested agent (x-axis), the response of each of six beads is shown (y-axis). Response is given as the median fluorescent intensity (MFI) at z-axis. Dotted bars indicate the probe of BA-1-coated bead; grid bars correspond to BA-2; right-slash bars are Yp-2; straight bars are Bru-2; right-dot bars indicate Ft-2; zigzag bars are Bp-2. From the y-axis, B is the blank blocks; Ba is Bacillus anthracis Sterne; Yp is Yersinia pestis EV76; Bru is Brucella spp. M5; Ft is Francisella tularensis and Bp is Burkholderia pseudomallei. The six bacterial coated beads are from species Bacillus anthracis, Yersinia pestis, Brucella abortus, Francisella tularensis, and Burkholderia pseudomallei, respectively. Each sample yields an appropriate response from each of the six beads present.

Mentions: Twenty-eight reference strains of certain bacterial species were tested for evaluation of the specificity of the two arrays. Table 3 indicated there are cross reactions existed among the same genus for universal primer PCR-based array. Such as it was positive signals for Bacillus thuringiensis, Bacillus cereus samples using the probes specific to Bacillus anthracis. Whereas there is no false-positive result or cross reactivity observed in multiplex PCR-based assay. Figure 4 shows the specificity of multiplex PCR array in a 3D axis with a matrix of each combination of four bacteria species by each multiplex array bead. The high MFI column indicated that each bead was only positive to its corresponding bacterium but not the other four bacteria.


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)

Specificity test of probes in multiplex primer PCR-coupled suspension array. For each tested agent (x-axis), the response of each of six beads is shown (y-axis). Response is given as the median fluorescent intensity (MFI) at z-axis. Dotted bars indicate the probe of BA-1-coated bead; grid bars correspond to BA-2; right-slash bars are Yp-2; straight bars are Bru-2; right-dot bars indicate Ft-2; zigzag bars are Bp-2. From the y-axis, B is the blank blocks; Ba is Bacillus anthracis Sterne; Yp is Yersinia pestis EV76; Bru is Brucella spp. M5; Ft is Francisella tularensis and Bp is Burkholderia pseudomallei. The six bacterial coated beads are from species Bacillus anthracis, Yersinia pestis, Brucella abortus, Francisella tularensis, and Burkholderia pseudomallei, respectively. Each sample yields an appropriate response from each of the six beads present.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368695&req=5

fig4: Specificity test of probes in multiplex primer PCR-coupled suspension array. For each tested agent (x-axis), the response of each of six beads is shown (y-axis). Response is given as the median fluorescent intensity (MFI) at z-axis. Dotted bars indicate the probe of BA-1-coated bead; grid bars correspond to BA-2; right-slash bars are Yp-2; straight bars are Bru-2; right-dot bars indicate Ft-2; zigzag bars are Bp-2. From the y-axis, B is the blank blocks; Ba is Bacillus anthracis Sterne; Yp is Yersinia pestis EV76; Bru is Brucella spp. M5; Ft is Francisella tularensis and Bp is Burkholderia pseudomallei. The six bacterial coated beads are from species Bacillus anthracis, Yersinia pestis, Brucella abortus, Francisella tularensis, and Burkholderia pseudomallei, respectively. Each sample yields an appropriate response from each of the six beads present.
Mentions: Twenty-eight reference strains of certain bacterial species were tested for evaluation of the specificity of the two arrays. Table 3 indicated there are cross reactions existed among the same genus for universal primer PCR-based array. Such as it was positive signals for Bacillus thuringiensis, Bacillus cereus samples using the probes specific to Bacillus anthracis. Whereas there is no false-positive result or cross reactivity observed in multiplex PCR-based assay. Figure 4 shows the specificity of multiplex PCR array in a 3D axis with a matrix of each combination of four bacteria species by each multiplex array bead. The high MFI column indicated that each bead was only positive to its corresponding bacterium but not the other four bacteria.

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