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Lucigen Details Polymerase Tailored for LAMP Assays; Potential in MDx Tests

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Genomics tools provider Lucigen has developed an isothermal amplification polymerase ideally suited for use in loop-mediated isothermal amplification-based assays.

The firm has also demonstrated how the polymerase can be used in LAMP-based diagnostic assays for various hemorrhagic fevers, influenza, and Clostridium difficile, and is currently seeking licensing or commercialization partners for the polymerase.

In a corporate-sponsored workshop held in conjunction with last week's Association for Molecular Pathology meeting in Phoenix, Ariz., Lucigen officials detailed their polymerase and discussed how it can be used to detect RNA and DNA targets using LAMP.

Lucigen's polymerase, called OmniAmp, combines thermostable strand displacement activity for LAMP-based amplification with reverse transcriptase activity to amplify directly from RNA, company officials said at the workshop.

OmniAmp can amplify either RNA or DNA targets in a single-tube, single-buffer system, providing much faster time to result than PCR. Several other isothermal amplification methods exist but rely on complex protocols, multiple enzymes, or special reagents to perform RNA-dependent amplification.

Lucigen, based in Middleton, Wis., has worked with Galveston National Laboratories to demonstrate the feasibility of a field-based nucleic acid testing system using the method for rapid detection of RNA from viral hemorrhagic fever agents such as Ebola, Marburg, and Lassa viruses.

The assays use the single-enzyme isothermal amplification method and lateral flow detection in a cassette-based system, and can detect target RNA in 5 to 20 minutes. Lucigen has verified the assays externally using nucleic acids extracted from cultured virus and from residual samples obtained from experimentally infected animal subjects at Galveston National Laboratories.

The company has also conducted in-house studies to demonstrate proof of principle for an OmniAmp- and LAMP-based Clostridium difficile assay.

For this assay, company scientists designed LAMP primers to target a conserved region at the 5' end of the tcdA gene of C. difficile. The primers were mixed with an isothermal buffer, polymerase, and the target DNA, followed by isothermal amplification. Conditions for amplification consisted of an initial denaturation step of five minutes followed by incubation for 40 minutes under isothermal conditions. The researchers also used SYBR Green dye in the reaction mixture to enable real-time monitoring of amplification products and post-reaction melt curve analysis.

Lucigen tested its assay on residual frozen stool samples from 13 patients presenting with C. difficile infection, comparing it to an unspecified method with US Food and Drug Administration approval, and found that its assay was able to amplify the tcdA target in less than 40 minutes and with comparable results to the FDA-approved test.

Officials noted at the AMP workshop that the company has also used its method to develop and demonstrate proof of principle for an influenza assay, an endeavor for which the company garnered a $2.8 million Small Business Innovation Research grant from the National Institutes of Health.

Curtis Knox, vice president of marketing and sales for Lucigen, told PCR Insider after the company's presentation that OmniAmp is "the same base enzyme" as 3173 Pol, a thermostable viral DNA polymerase that was isolated from hot springs and can enable true one-step, single-enzyme, reverse transcriptase PCR.

Knox noted that the company has developed "a few different modifications" of 3173 Pol, including OmniAmp, and that the company is looking to collaborate with or license the enzyme to commercial partners interested in developing LAMP-based infectious disease diagnostics. The company has been awarded or applied for multiple patents on its various enzymes.

Lucigen is fully licensed to provide LAMP reagents for research use under an agreement with Eiken Chemical, which owns the LAMP patents. However, OmniAmp and other enzymes from Lucigen may not be used for LAMP-based human or diagnostic purposes without obtaining a license from Eiken.

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