In Real Time
“It gets complicated when you’re doing multiplexing,” comments Dr. Tebbs. “If it’s only one reaction, it’s only a couple of primers. When you start adding other reactions, you’ve got more possibility of interference. The primers can interact. The probes can interact. The effort is trying to mitigate those interaction effects and balance out the different reactions.”
In the food pathogen detection business, there are specific regulatory requirements for which pathogens should be detected and which should not. One common problem is the detection of “nearest neighbor” organisms that are not actually target organisms themselves. For example, Vibrio mimicus looks a lot like cholera (hence, the name mimicus). There is zero tolerance for cholera in the food supply, but the presence of V. mimicus could cause false positives for cholera.
The payoff for all this trouble is an assay of surpassing utility. “One of the big advantages of doing these multiplexes is that you get to detect and assay many different organisms at once. It takes less time to do your work.
“Using classical methods, if you look at each one separately,” Dr. Tebbs points out, “it takes two or three days to determine if an organism is present.” In the food business this is often two or three days too long, and many people may become ill before the pathogen is found.
Another interesting application of PCR to multiplex biodetection is the use of nested multiplex PCR. In this strategy, PCR is carried out in two stages. The first stage uses universal amplification, like the BEADS assay previously mentioned. The PCR products are then diluted and moved into an array of reaction chambers for a second stage of PCR using specific primers targeted to a much shorter sequence.
Thus, each final reaction product has been created using four primers instead of the usual two, with the second set “nested” inside the first set. This circumvents the problem of washout by the dominant species, so that minority species can be detected.
One big problem with nested multiplex PCR, however, is contamination of the amplicon. Idaho Technology (www.idahotech.com) has a solution to this problem. The reaction is carried out in a completely sealed chemical circuit board. It actually resembles a glorified piece of bubble wrap, but in this case each bubble is a chamber that contains a stage of the assay, beginning with sample prep all the way through a 120-plex second-stage PCR.
The instrument that shepherds the samples through these plastic chambers is called the FilmArray™Instrument. The technology came from government field testing but it’s being developed at Idaho for medical testing. Multiplex pathogen detection will, for the first time, allow physicians to diagnose complex, multiple infections right at the bedside (in about 55 minutes), according to Idaho. These types of infections are much more prevalent than once thought, and if they can be detected in the early stages, it may be possible to prevent complications.
The FilmArray is able to detect pathogens at concentrations ranging from 10,000 cfu/mL down to 5 cfu/mL in the same sample from up to 120 samples simultaneously, reports the company. The system includes a software analysis package developed in-house at Idaho Technology.