[Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria].
OBJECTIVE
To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria.


METHODS
16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed.


RESULTS
After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples.


CONCLUSIONS
The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.
Institute of Health Quarantine, China Academy of Inspection and Quarantine, Beijing, China.
Hai-yan
HY
Wen
Jing
J
Wang
Heng-chuan
HC
Liu
Xiao-hong
XH
Sun
Yu
Y
Yang
Kong-xin
KX
Hu
Lin-jun
LJ
Shan
D001408:Bacillus anthracis / Q000302:isolation & purification*; D023482:Bioterrorism / Q000517:prevention & control; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005604:Francisella tularensis / Q000302:isolation & purification*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015010:Yersinia pestis / Q000302:isolation & purification*
D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S