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Seegene Develops Highly Multiplexed Quantitative Real-Time PCR Chemistry, HPV Genotyping Assay

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Molecular diagnostics and assay technology developer Seegene said this week that it has added yet another multiplex PCR-based chemistry to its portfolio of nucleic acid amplification, detection, and quantification technologies.

Currently referred to as "multiplex quantification" chemistry, the technology enables real-time PCR-based detection and quantification of up to 20 analytes from a single sample and in a single tube using existing multiplex-enabled thermal cyclers.

In addition, Seegene is using the technology to develop a highly multiplexed, two-tube assay to simultaneously detect, differentiate, and quantify 19 high-risk and nine low-risk human papillomavirus genotypes.

"This is a totally new approach [that] we discovered … a few months ago," Jong-Yoon Chun, founder, CTO, and CEO of Seegene, told PCR Insider this week. "We have never introduced this technology before, or even disclosed it to other companies. As far as I know, multiplex quantification based on real-time PCR has not been introduced previously [in general]."

Indeed, the multiplexing limit of current commercial quantitative real-time PCR assays is around five or six targets due to spectral overlap of fluorescent probes.

Many researchers in industry and academia have been pushing the boundaries of multiplex real-time PCR. For instance, as reported last week in PCR Insider, researchers from the National University of Ireland Galway have developed and validated a multiplex real-time PCR assay that can identify 10 targets of the Mycobacterium tuberculosis complex (PCR Insider, 5/24/2012).

But, like most other similar assays, the NUI-Galway group's test is limited by current real-time PCR instrumentation, requiring two separate five-plex steps to achieve its results.

Seegene, which is based in Seoul, Korea, with a US office in Gaithersburg, Md., has for the past several years been developing a plethora of chemistries in a bid to break down the barriers of multiplexed real-time PCR, but has similarly been limited by commercial instrumentation platforms.

For instance, Seegene based most of its early commercial activities around a pair of flagship technologies: dual-priming oligonucleotide, or DPO; and real amplicon detection, or READ. DPO is designed to generate consistently high specificity by blocking extension of non-specially primed templates and eliminating primer competition, while READ combines the advantages of multiplex and nested real-time PCR while increasing specificity and sensitivity 10- to 100-fold over other real-time PCR technologies.

Then, almost a year ago, at the 2011 AACC annual meeting, Seegene demonstrated for the first time its previous 'new' multiplexing technology, called TOCE, which enables detection of as many as 16 targets via melt curve analysis using a single fluorescence channel on most commercial real-time PCR instruments (PCR Insider, 7/21/2011).

At the time, David Dolinger, Seegene's vice president of business development, said that all of these assay chemistries were constrained by current instrumentation platforms, noting that this has limited Seegene in how it designs its assays, and had prompted the company to adapt current commercial instruments to its chemistry "instead of trying to wait for someone to come up with a 16-channel real-time instrument."

It appears that Seegene has now taken the next incremental step in this process. Unlike the TOCE technology, which offers a high degree of multiplexing but is not quantitative, the company's new "multiplex quantification" technology is designed to simultaneously detect and quantify up to 20 analytes using a four-channel real-time PCR instrument.

"Any existing multiple-channel instrument can be applied," Chun said this week. Currently, the company is validating the new technology on the Life Technologies ABI 7500 and Bio-Rad CFX 96 systems, he added.

In order to demonstrate the clinical potential of its technology, Seegene has already used it to develop a highly multiplexed real-time PCR-based assay to detect, differentiate, and quantify 19 high-risk and nine low-risk HPV genotypes. Because the assay has 28 total targets, it is divided into two separate reaction tubes, which wouldn't drastically affect the cost or complexity of performing the test, Chun said.

Chun also said that the new technology has the potential to drastically reduce the cost of multiplex real-time PCR to under $10 an assay, depending on the number of targets present.

Seegene's goal is to disseminate its technologies, including the new multiplex quantification chemistry, as widely as possible through licensing deals, company partnerships, and OEM agreements. The company has always been open to such partnerships, but has in the past put more resources into developing its own diagnostic products, several of which are sold outside the US but none of which has been cleared in the US.

Just last week, Seegene announced that it was partnering with DuPont Nutrition & Health to jointly develop highly multiplexed assays to detect foodborne pathogens by combining Seegene's DPO and TOCE technologies with DuPont's benchtop BAX System (PCR Insider, 5/24/2012).

And, in an effort to drum up interest in the new technology, the company will unveil both it and the HPV test in July at the American Association for Clinical Chemistry meeting in Los Angeles. Chun said that he expects a commercial version of the assay to be ready by September or October of this year and added that the HPV test will be just one demonstration of the new technology.

"Why are people not applying molecular diagnostics? Because the systems are not available, or are very expensive," Chun said. "We are looking to make it so people can apply it on a routine basis. I call this a sleeping market. In order to wake it up, it's better to share our technology than to develop it alone."

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