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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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Gel electrophoresis (1% agarose gel) of the amplification products by universal primers PCR. Lane 1. Francisella tularensis; Lane 2. Burkholderia pseudomallei; Lane 3. Yersinia pestis EV76; Lane 4. Brucella spp. M5; Lane 5. Bacillus anthracis Sterne; M: DL2000 DNA Marker.
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fig1: Gel electrophoresis (1% agarose gel) of the amplification products by universal primers PCR. Lane 1. Francisella tularensis; Lane 2. Burkholderia pseudomallei; Lane 3. Yersinia pestis EV76; Lane 4. Brucella spp. M5; Lane 5. Bacillus anthracis Sterne; M: DL2000 DNA Marker.

Mentions: In this assay, 16 s rDNA was amplified by the average size of 250 bp as Figure 1 indicated the gel electrophoresis with universal primer PCR amplification. The multiplex PCR factors have been optimized to approach the best reaction condition. The optimum reaction mixture contained 30 μL of of the master mix, 80 pmol of primer FT-F, FT-R, BP-F, BP-R, BA-1-F, BA-1-R, 100 pmol of primer BA-2-F, BA-2-R, YP-F, YP-R, 120 pmol of primer Bru-F, Bru-R each, 2 μL of DNA template. Thermal cycles included 1 cycle of 95°C for 10 min, 32 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, followed by a final extension of 7 min at 72°C. Figure 2 shows the gel electrophoresis the multiplex PCR products.


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)

Gel electrophoresis (1% agarose gel) of the amplification products by universal primers PCR. Lane 1. Francisella tularensis; Lane 2. Burkholderia pseudomallei; Lane 3. Yersinia pestis EV76; Lane 4. Brucella spp. M5; Lane 5. Bacillus anthracis Sterne; M: DL2000 DNA Marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Gel electrophoresis (1% agarose gel) of the amplification products by universal primers PCR. Lane 1. Francisella tularensis; Lane 2. Burkholderia pseudomallei; Lane 3. Yersinia pestis EV76; Lane 4. Brucella spp. M5; Lane 5. Bacillus anthracis Sterne; M: DL2000 DNA Marker.
Mentions: In this assay, 16 s rDNA was amplified by the average size of 250 bp as Figure 1 indicated the gel electrophoresis with universal primer PCR amplification. The multiplex PCR factors have been optimized to approach the best reaction condition. The optimum reaction mixture contained 30 μL of of the master mix, 80 pmol of primer FT-F, FT-R, BP-F, BP-R, BA-1-F, BA-1-R, 100 pmol of primer BA-2-F, BA-2-R, YP-F, YP-R, 120 pmol of primer Bru-F, Bru-R each, 2 μL of DNA template. Thermal cycles included 1 cycle of 95°C for 10 min, 32 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, followed by a final extension of 7 min at 72°C. Figure 2 shows the gel electrophoresis the multiplex PCR products.

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