Researchers from the UK's Defence Science and Technology Laboratory have demonstrated the feasibility of using Life Technologies' microfluidic TaqMan Array Cards to detect multiple bacterial agents with the potential to cause acute disease and be used in a bioterror attack.
The work builds on previous research from a different team that showed how TaqMan Array Cards could be used to detect multiple respiratory pathogens from clinical samples, thus demonstrating the potential of the cards — currently intended for research use only — to be used in a clinical diagnostic capacity.
However, although the DSTL team demonstrated that the architecture of the microfluidic cards could be a boon for higher throughput screening of potential biological agents, they also discovered that in general the card-based assays had lower sensitivity than standard singleplex real-time PCR — a fact they said was likely due to sample prep methods and could be improved upon.
Simon Weller, a group technical leader for genetic detection at DSTL, told PCR Insider this week that his laboratory decided to evaluate the TaqMan Array Cards as part of its effort to test new technology platforms that could detect as many biological agents as possible for the purposes of bioterror screening.
This is in contrast to currently used molecular methods such as single-plex real-time PCR that, while highly sensitive, is still relatively limited in its ability to rapidly screen for multiple bacterial agents and is "logistically burdensome," Weller said.
"We needed to extend that capability, because the technology has been extended," he said. "You've got to move with the times, as it were, and we are looking at a variety of platforms, actually," Weller said, although he declined to identify which ones due to the sensitive nature of the work
"We have quite a good relationship with Life Tech, and … their [sales] rep said [that] we could use [TaqMan array cards] for this," Weller said. The array cards, he noted, were not developed for pathogen detection, but for gene expression studies, "but [Life Tech] said let's see if you can get your assays onto our cards, and we'll take it from there."
The results of their efforts were published in a paper last month in PLoS One.
In short, Weller and colleagues took ten previously developed real-time PCR assays for five agents — Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis — and worked with Life Tech scientists to port them to custom-made 384-well TaqMan Array Cards, which are designed for one to eight samples to be run in parallel against 12 to 384 TaqMan assays in individual 1-µL reaction chambers.
The custom cards for the DSTL research comprised eight channels of 24 individual PCR reactions, with each reaction comprising two replicates, thus providing 48 individual reaction chambers per sample. The researchers pipette-loaded each of the reaction chambers with 50 µL of DNA extracted from the five biological agents in question and 50 µL of TaqMan master mix from Life Tech; and they ran the cards on Life Tech's ViiA 7 real-time PCR system.
They compared the ability of these assays to detect varying amounts of DNA from these five biological agents with that of single-plex real-time PCR using the same primers, probes, and master mix, and run on Life Tech's 7500 Real-Time PCR System.
In general, the DSTL scientists determined that the single-plex PCR assays were about a log more sensitive than those run on the TaqMan array cards, with the single-plex format detecting nearly 100 percent of the targets when 100 femtograms of target DNA was added to the reactions.
In comparison, using the array cards, not all of the agent PCRs responded even when 500 femtograms of DNA was added to a channel on the card. "However, the presence of multiple replicates to agent PCRs increased the chances of agent detection, where agent detection is defined as at least one agent PCR replicate in a channel returned a positive result," the researchers wrote.
Using this definition, the TaqMan array cards detected all agents 100 percent of the time when 500 femtograms of DNA was added to each channel and seven replicates were used; while at the 100-femtogram level, and 14 replicates, Y. pestis was detected 100 percent of the time; B. mallei and F. tularensis 93 percent; B. anthracis 71 percent; and B. pseudomallei 43 percent.
"You're sacrificing a bit of sensitivity," Weller said. "But when we got the array cards, honestly, I didn't think they'd work as well as they did. I thought they'd probably get down to picogram [levels of] detection, and that would be it really. And we needed to work out whether we could live with that. What we've shown is they probably go below even the picogram level."
In addition, Weller said, the group deduced that the lower sensitivity was likely not the fault of the array cards, per se, at least the current versions of the cards, which were designed for gene expression studies and not pathogen detection.
"[Life Tech] is expecting you to put in … nanograms of cDNA because it's a gene expression study," he said. "Whereas for pathogen detection, you aren't putting in nanograms, you're putting in a sample, where there might not be nanograms of the thing you're looking for. You've got to accept that and look at what you're actually doing rather than follow the [Life Tech] gene-expression protocol."
Weller noted that due to the microfluidic architecture of the array cards, not all of the added sample may be getting into the designated reaction volume areas. "These are 1-µL reaction chambers. But you're actually putting into each channel 100 µL," Weller said. "So you can tell that a lot of sample doesn't get analyzed by anything, it gets trapped in the microfluidics. If the manufacturers made the reaction chambers a bit bigger in terms of volume, that could actually increase the sensitivity, because you're getting more of the sample into each of the reaction chambers."
Further, Weller believes that sample prep methods play a huge role, a theory that he said is backed up by the results of a similar pathogen-detection study published last year in the Journal of Clinical Microbiology by researchers from the US Centers for Disease Control and Prevention, who investigated the use of the TaqMan Array Cards to detect 21 respiratory pathogens from 292 clinical respiratory samples.
In that study, the CDC researchers found that the array card assays achieved an overall sensitivity of 89 percent with 98 percent specificity when compared to individual real-time PCR assays.
"You need to optimize everything else, such as DNA extraction," Weller said. "We tested some clinical samples on the array cards using a standard sort of Qiagen spin column method, and the results were a little bit patchy. We're pretty sure that's because of the DNA extraction method rather than the array cards."
"If you look at the [CDC] paper, they … used a magnetic bead DNA extraction method, and they bridged the sensitivity gap," he added. "We both talk about a log difference in sensitivity when testing straight DNA, but then they tested actual real-world samples using the magnetic bead DNA extraction method, and they got really good results."
Despite the need to tweak the array cards to increase assay sensitivity, Weller said that their potential benefit as a multiplexed screening method may outweigh any drawbacks.
"You can increase coverage of potential threats," he said. "And you can also increase the confidence in your identification, because you can have more than one PCR [reaction] per agent, and that increases your confidence."
Further, because each reaction chamber holds just one PCR reaction, "that means you can swap assays in and out pretty easily," Weller said. "In a multiplex PCR, where all the reactions are in one reaction [tube], if you swap one assay in or out you have to make sure all the other assays are still working as they were before. With the array cards it doesn’t matter, because each assay is one reaction chamber on its own. That makes them pretty flexible."
In an e-mail to PCR Insider, Jon Sherlock, market development manager at Life Tech, noted that this spatial multiplexing is a "key advantage" for the array card format.
Sherlock also said that other customers, like the UK's Health Protection Agency and the National Institute for Biological Standards and Controls, have been using the cards for pathogen surveillance DNA work.
Life Tech, he said, "offers a spectrum of research-use products in this format, including DNA and microRNA applications," but also "encourages researchers and assay developers to work with us to create custom products in this format." In addition, he said that the company "is constantly working on improvements for ease of use and overall performance," and that sample prep and workflow improvements, such as using the company's PreAmp master mix to increase sensitivity, are in the works for the TaqMan arrays.