Advanced Techniques in Diagnostic Microbiology

482 J. Han
suspension hybridization reaction mixture to a dual-laser detection device. A red
laser identifies each bead by its color-coding, and a green laser detects the hybridization
signal associated with each bead. Software is used to collect the data
and report the results in a matter of seconds.
The platform is specific because only the probes that are captured by the beads
are recognized by the green laser as signal. Any signal not associated with a specific
set of color-coded beads is considered background. The platform is also very sensitive.
Each bead has as many as 10 8 COOH groups on its surface for linking capture
oligos. The green laser can detect the signal for as few as eight fluorescently labeled
probes that are captured by a bead. Another important feature of the xMAP platform
is its repeatability. Because everything occurs in a homogeneous solution (from
bead manufacture, color-code staining, and capture probe coupling to product hybridization
and data collection), highly repeatable results are obtained with this
platform. The xMAP method for collecting and reporting data also contributes to repeatability.
Typically, there are 5000 beads added per reaction for each color-coded
bead set. Each bead set is specific for a particular disease marker, such as a mutation
or a pathogen. The laser counts 100 microspheres from each bead set and reports
the median fluorescent intensity (MFI). Thus, the data represents 100 microbeadassociated
data points, not just one data point produced by a standard array.
Sequencing-based methods and platforms provide the best specificity of all nucleic
acid detection methods. However, abundant, pure templates must be generated
first. The costs associated with reagents, instruments, and labor are relatively high.
Sequencing is an essential technology for research laboratories, but it is usually
too complicated and cost-prohibitive for routine clinical applications.
Examples of Technology Integration
It is not expected that one company, institution, or individual should develop a
complete solution for all components of a molecular diagnostic process. Instead,
it is more favorable for the best available methods and platforms to be combined,
along with individual creativity, to realize the potential of molecular differential
diagnosis. In the following section (see Table 27.4), there will be an evaluation
of several companies that have created multiplex technologies and products with
different strategies for technology integration, and there will be a discussion of the
advantages and disadvantages of these strategies.
TABLE 27.4. Comparison of different methods/platform integration strategies.
Amplification Detection Multiplex Post-PCR Posthybridization
Company method/platform method/platform capability clean-up washes
Genaco Templex Luminex xMAP Yes No No
TM Bioscience PCR/TM100 Luminex xMAP Yes Yes Yes
Roche Real-time PCR Light Cycler Limited No No
Fluidigm Real-time PCR IFCs Yes No No
Prodessa PCR ELISA Yes/No Yes Yes
Maxim Biotech PCR Gel electrophoresis Yes No No