Rapid oligonucleotide suspension array-based multiplex detection of bacterial pathogens.
A gene-specific microsphere suspension array coupled with 15-plex polymerase chain reaction (PCR) was developed to screen bacterial samples rapidly for 10 strains of bacteria: Shigella spp. (S. flexneri, S. dysenteriae, and S. sonnei), Staphylococcus aureus, Vibrio cholerae (serology O1 and O139), Legionella pneumophila, and Clostridium botulinum (types A, B, and E). Fifteen sets of highly validated primers were chosen to amplify target genes simultaneously. Corresponding oligonucleotide probes directly conjugated with microsphere sets were used to specifically identify PCR amplicons. Sensitivity tests revealed that the array coupled with single PCR was able to detect purified genomic DNA at concentrations as low as 10 copies/μL, while the multiplex detection limit was 10-10⁴ copies/μL. The assay was validated using water samples artificially spiked with S. aureus and S. dysenteriae, as well as water specimens from swimming pools previously identified to contain S. aureus.
1 State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences , Beijing, China .
Jinyin
J
Zhao
Lin
L
Kang
Rui
R
Hu
Shan
S
Gao
Wenwen
W
Xin
Weijun
W
Chen
Jinglin
J
Wang
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D001906:Botulism / Q000175:diagnosis* / Q000382:microbiology; D002771:Cholera / Q000175:diagnosis / Q000382:microbiology; D003014:Clostridium botulinum / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004405:Dysentery, Bacillary / Q000175:diagnosis* / Q000382:microbiology; D016952:Legionella pneumophila / Q000235:genetics / Q000302:isolation & purification; D007877:Legionnaires' Disease / Q000175:diagnosis* / Q000382:microbiology; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification; D014734:Vibrio cholerae / Q000235:genetics / Q000302:isolation & purification; D014871:Water Microbiology
D017931:DNA Primers; D004269:DNA, Bacterial