ABSTRACT
We have developed a 10-plexed PCR assay coupled to a 12-plexed liquid bead array to rapidly screen environmental samples for B. anthracis, Y. pestis, F. tularensis, and B. melitensis. Highly validated species-specific primer sets were used to simultaneously amplify multiple diagnostic regions unique to each individual pathogen. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The hybridized beads were processed through a flow cytometer, which detected presence and quantity of each PCR product. The assay was optimized to allow for maximum sensitivity in a multiplexed format. A high-throughput demonstration was performed where 384 simulated environmental samples were spiked with different amounts of B. thuringensis spores and pathogen DNA. The samples were robotically processed to extract DNA and arrayed for multiplexed PCR-liquid bead detection. The assay correctly identified the presence or absence of each pathogen and collected over 3000 individual data points within a single 8-h shift for approximately $4.00 material costs per environmental sample in a 10-plexed assay.
Subject(s)
Bacillus anthracis/genetics , Brucella melitensis/genetics , DNA, Bacterial/genetics , Francisella tularensis/genetics , Yersinia pestis/genetics , Bacillus anthracis/isolation & purification , Bioterrorism , Brucella melitensis/isolation & purification , DNA Primers , DNA Probes/genetics , DNA, Bacterial/isolation & purification , Francisella tularensis/isolation & purification , Microspheres , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Spores, Bacterial/genetics , Spores, Bacterial/isolation & purification , Yersinia pestis/isolation & purificationABSTRACT
Ribosomal DNA sequence analysis, originally conceived as a way to provide a universal phylogeny for life forms, has proven useful in many areas of biological research. Some of the most promising applications of this approach are presently limited by the rate at which sequences can be analyzed. As a step toward overcoming this limitation, we have investigated the use of photolithography chip technology to perform sequence analyses on amplified small-subunit rRNA genes. The GeneChip (Affymetrix Corporation) contained 31,179 20-mer oligonucleotides that were complementary to a subalignment of sequences in the Ribosomal Database Project (RDP) (B. L. Maidak et al., Nucleic Acids Res. 29:173-174, 2001). The chip and standard Affymetrix software were able to correctly match small-subunit ribosomal DNA amplicons with the corresponding sequences in the RDP database for 15 of 17 bacterial species grown in pure culture. When bacteria collected from an air sample were tested, the method compared favorably with cloning and sequencing amplicons in determining the presence of phylogenetic groups. However, the method could not resolve the individual sequences comprising a complex mixed sample. Given these results and the potential for future enhancement of this technology, it may become widely useful.
