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Researchers from U of Nebraska, Streck Develop Rapid Sample Prep, PCR Method for C. Diff Testing

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Researchers from clinical lab supply company Streck, the University of Nebraska, Lincoln, and the UN Medical Center have developed a method for extracting and detecting Clostridium difficile DNA from stool samples more rapidly than existing commercial methods and with comparable accuracy.

The protocol combines a novel, prototype single-use "lysis microreactor" with Streck's commercially available Philisa thermal cycler, an ultrafast endpoint PCR system that can produce results in less than 15 minutes.

In addition, the researchers believe that the combined technologies could serve as an inexpensive, rapid point-of-care system for diagnosing a variety of infectious diseases, particularly if Streck develops a real-time version of its thermal cycler — an endeavor that the firm, which has not previously explored the molecular diagnostics market, is currently evaluating, a company official said this week.

"We are definitely evaluating the potential of doing that and seeing if it fits into Streck's business model with the next [PCR] instrument," Matthew Kreifels, molecular technology project manager at Streck, told PCR Insider this week.

Kreifels noted that while Streck has not traditionally developed clinical or molecular diagnostic products per se, the company does have a good deal of experience in certain clinical markets.

"That's not clinical diagnostics, but they are used in the clinical market — they're IVD," he said. "So we have a lot of experience bringing those types of controls through [the IVD approval process]. Do we understand that there is a different level of scrutiny for developing a clinical diagnostic for the molecular market? We do, and we are looking at how we might restructure ourselves differently to do that."

In a study funded by Streck and published last month in the Journal of Molecular Diagnostics, the researchers described their prototype sample prep device, a lysis microreactor, or LMR, featuring dry chelating resin beads from Bio-Rad Laboratories and a hydrophobic polystyrene capture strip attached to a removable cap.

The researchers also described their protocol for using the microreactor to extract DNA from stool samples with minimal user intervention: The stool sample is mixed with lysis buffer, transferred to the cartridge, heated to 92° C, and mixed for five minutes to lyse bacterial cells, denature DNA, and enable single-stranded DNA to bind to the capture strip.

Then, the polystyrene strip is removed and washed, placed in a PCR tube with master mix, and placed into Streck's Philisa thermal cycler for rapid PCR amplification and subsequent detection via gel electrophoresis. The entire process, minus the electrophoretic analysis step, can be accomplished in under 20 minutes, according to the researchers.

The scientists tested their protocol against existing methods for diagnosing C. difficile infections. Because there is no accepted gold standard for such testing, they compared their LMR-PCR method to a protocol routinely used in the UNMC clinical laboratory: a two-step diagnostic algorithm in which samples found positive for glutamate dehydrogenase (GDH) and negative for toxin using a dual-enzyme immunoassay are reflexed for testing with Meridian Bioscience's Illumigene C. difficile loop-mediated isothermal amplification-based test.

Specifically, they used both methods to examine 198 stool samples from patients with suspected C. difficile infection. Of these samples, 48 were positive for both GDH and toxin, and 81 were negative for both when tested using the immunoassay. Subsequent testing using the LMR-PCR method resulted in perfect correlation with these results.

Of the 69 enzyme immunoassay discordant samples that were tested with both the reflex Illumigene assay and the new method, 64 were in agreement. Of these, 29 were positive and 35 were negative by both PCR tests; and the remaining five samples were positive for C. difficile using the new method but negative using Illumigene.

Overall, the researchers noted, LMR-PCR testing matched enzyme immunoassay/Illumigene reflex in 193 of 198, or 97.5 percent of samples.

While the sensitivity of the LMR-PCR method was comparable to standard methods, the researchers noted that the potential high speed and low cost of their protocol make it promising for use as a point-of-care testing system for C. difficile or even other infectious diseases.

"The current LMR is an individual temperature-controlled unit for a single sample; however, it would be straightforward to expand it to an eight-well device to match the eight sample positions in the Philisa [thermal cycler]," the researchers wrote in their paper. "With the addition of real-time PCR, the throughput of the system could be as high as 192 samples in an 8-hour workday."

Diving into MDx?

Streck, based in Omaha, Neb., has for the last several years taken advantage of innovation at nearby academic institutions and medical centers such as UN-Lincoln and UNMC by developing various products based on technologies from those institutions' laboratories.

Chief among these products is the Philisa, an ultrafast thermal cycler that Streck launched in September of last year (PCR Insider, 9/8/2011). The company claims that the system can reduce PCR amplification time to less than 15 minutes using standard chemistries by offering heating ramp rates of up to 15°C/s and cooling ramp rates of up to 12°C/s.

The Philisa performs "true PCR, and our definition of that is no special additives or chemicals are needed," Streck's Kreifels told PCR Insider.

"Along with the thermal cycler, we developed a proprietary plastic tube [with] a sample volume advantage compared to other PCR companies," he added. "We're basically able to handle 10 to 50 microliters. Generally, the people who are going faster are doing much smaller volumes, and they're doing it on a plate, and that's not what we're doing. From a speed perspective, we've had really good success with reducing very long protocols, typically 3-hour protocols, by 50 to 70 percent."

The company highlighted a forensic science example of this in a recent white paper. Specifically, they demonstrated that the Philisa could generate accurate short tandem repeat, or STR, amplicons using standard primer sets from Life Technologies' Applied Biosystems AmpFℓSTR Identifiler kit in less than 17 minutes. In comparison, the amplification step in the human DNA identification process usually takes around three hours, the authors of the white paper noted.

Kreifels said that he could not comment on Streck's specific R&D or business development initiatives, including whether the company is engineering or plans to engineer a real-time version of the Philisa, or whether it will develop a commercial product based on the LMR cartridge described in the JMD paper.

However, he echoed the researchers' claims that the LMR-PCR solution has the potential to change the way molecular infectious disease testing is performed, particularly from difficult-to-work-with sample types like stool.

"Currently in the market, there are other mechanical methods, beads, for doing the same thing. But it takes a lot of time. Cepheid certainly has a model, but they're not the cheapest product on the market," Kreifels said.

"The whole basis of this is that you … want to handle stool as [little] as possible, and the [researchers] would prefer to be able to inexpensively reduce it down to DNA and capture that and transfer it for other purposes as rapidly as possible with the least amount of exposure to the technician," he added. "That's what they strove for in their prototype."

If Streck were to move forward with commercial product development, "we would try to fit a niche that would fill the same need as the conventional [Philisa] PCR platform … and try to reduce all those steps and go sample to result as fast and accurately as we can," Kreifels said.

"Even though [we haven't been] in the molecular field before … we're learning, and we want to do it right, and we're a company that, when we take on something, we don't take it lightly," he added.

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