charides (Schaudies, 2014). Most antibody-based identification methods (such as enzyme-linked immunosorbent assay, or ELISA) involve immobilizing antibodies for particular antigenic targets on solid substrates. These “capture” antibodies then bind to the antigens produced by the microorganismal cells. This binding is then detected with a second antibody to the same antigen that contains a “reporter molecule” that produces a detectable signal based on optical absorbance or fluorescence. Examples of antibody-based platforms include Luminex, PathSensors, Inc., and TacBioHawk. (See Schaudies, 2014, for a more in-depth review.) The most notable advantage of antibody- or protein-based detection systems is their speed—many of them can provide answers in 3 to 10 minutes. Some of the systems can reach sensitivities approaching that of nucleic acid amplification techniques, such as PCR-based assays. However, for microbial forensic purposes, they often lack sufficient specificity to discriminate beyond the level of species. Platforms that perform protein and antibody/antigen detection, such as electrochemiluminescence (ECL), are also needed to detect protein toxins, such as ricin and botulinum. Schaudies (2014:175) states that antibody-based systems are “more effective and consistent than nucleic-acid-based systems for the detection and identification of toxins.” This statement is based not on efficacy of an assay, but more on the degree of toxin purification; in some toxin preparations, the DNA that codes for production of the toxin may not be present or nucleic acid concentrations may be reduced below limits of detection. Toxin treatment also may degrade DNA from the sample. As with PCR, these platforms are only as good as the assays developed for the intended target. Knowledge of the target being pursued is necessary as are thoughtfully designed and validated assays to use ECL effectively.
Nucleic acid–based detection and identification technologies provide the ability to examine the genetic as well as structural information associated with a pathogen. Among the early tools developed to access an organism’s genetic information were nucleic acid amplification techniques, such as PCR. PCR allows specific fragments of genomic DNA to be isolated and their copy number amplified. Over time, variations of the basic PCR technique have developed, such as “multiplex PCR,” which allows several DNA genomic regions to be amplified in the same reaction set, and quantitative or “real-time” PCR (qPCR), which can measure the quantity of a target sequence in real time. Multiplex PCR technologies do have limitations. Most allow up to five different fluorophores to be detected simultaneously. Other platforms have attempted to increase this to as many as 20 fluorophores simultaneously, which would be a big advance in multiplex capability. Real-time qPCR is a rapid and sensitive nucleic acid signature detection technology that has proved effective in both clinical and microbial forensic applications. Some platforms