By Ben Butkus
Researchers from Lawrence Livermore National Laboratory have developed a disposable, point-of-care, qualitative testing device that enables extraction and amplification of target nucleic acids followed by colorimetric detection in a single tube, according to a recently published research paper.
LLNL is currently seeking industry partners to license and develop the device, which could enable ultra-fast and inexpensive infectious disease testing in the human and veterinary medicine markets.
The device was developed in the laboratory of Jane Bearinger, a senior scientist and medical technology program leader at LLNL, in an attempt to identify a point-of-care testing method with the sensitivity and specificity of PCR but a much lower cost.
According to a paper published this month in IEEE Transactions on Biomedical Engineering, the LLNL device integrates a single-tube nucleic acid extraction method with loop-mediated isothermal amplification or reverse transcriptase LAMP.
As described in the paper, the LLNL device uses a swab to collect sample from surfaces, oral or nasal cavities, or lesions; and then transfer it to a Whatman FTA card — a 4-mm disc of cellulose-based nucleic acid binding paper commonly used in forensics and other nucleic acid testing applications.
The FTA disc is then positioned in a sealable, disposable polypropylene containment tube with a manual loading port. Purification reagents are then added to the tube via syringe and waste materials pipette out via a septum at the bottom of the tube. This step is repeated, and then LAMP reagents, including the colorimetric dye, are loaded through the port so as to contact the cellulose disc containing the sample.
Next, the reagents are isothermally heated to 63°C for about an hour in a simple heated tube rack, thus enabling sequence-specific target nucleic acid amplification. If amplification occurs, it induces a visible color change of purple to blue in a colorimetric dye. According to the researchers, the entire process takes about 90 minutes.
In the IEEE paper, the researchers used their device to detect methicillin-resistant Staphylococcus aureus genomic DNA, as well as recombinant and live foot-and-mouth disease virus, a highly infectious viral disease of cloven-hoofed animals.
They were able to achieve PCR-level sensitivity and specificity — detecting about 17 genomic copies of MRSA and detecting FMDV diluted down to 10-5. The MRSA primers — the subject of a forthcoming publication — were designed using open-source LAMP primer design software and were purchased from BioSearch. The FMDV primers had been previously published.
Further advantages of the assay, according to its developers, include the fact that it is completely self-contained, reducing the risk of cross-contamination via laboratory testing equipment; and it does not require expensive and complex laboratory equipment, making it ideal for low-resource and remote settings. In fact, the only component of the system requiring power is the isothermal heating unit, which the researchers wrote "could be simplified and run via battery power."
In the current prototype of the device, the researchers note, purification reagent and buffer streams are introduced through the swab via syringe; reagents are introduced through the septum via pipette; and waste streams are evacuated through the septum.
"While such introduction and evacuation of solutions are appropriate in a laboratory setting, our planned next-generation prototype system will reduce platform complexity and facilitate field deployment by fully enclosing wash and reagent solutions and by fully containing waste streams," the researchers wrote.
Other alterations being considered by the group include adding an internal, positive control and an optional, inexpensive spectrometer to increase detection robustness.
The scientists said that current direct costs per disposable prototype are about $15, a figure that would likely significantly drop upon mass production.
In a solicitation published last week on FedBizOpps.com, LLNL's Industrial Partnerships Office noted that it is now offering the opportunity to license and develop the POC device, and is "seeking industry partners with a demonstrated ability to bring such inventions to the market."
The solicitation notes that the device may be particularly useful in POC human medicine and field veterinary applications; and that LLNL is currently pursuing patent protection for the device and assay developments.
Bearinger's LLNL lab has its hand in a variety of bioengineering projects that use chemical engineering and materials science approaches, including an inexpensive and rapid method of making protein nanoarrays for various proteomic applications, as reported in 2009 by PCR Insider sister newsletter ProteoMonitor.
The isothermal LAMP technique is gaining traction as a simple, inexpensive alternative to PCR for nucleic acid testing. Notably, Meridian Biosciences' FDA-cleared and CE Marked Illumigene assay, a LAMP-based assay for detecting C. difficile (PCR Insider, 7/22/10). In 2006, Meridian licensed the LAMP technology from Japan's Eiken Chemical for use in infectious disease testing.
In addition, OptiGene, LGC, and the University of Southampton, are developing an ultra-rapid molecular testing platform to diagnose sexually transmitted diseases, for which OptiGene has licensed the LAMP technology (see related story, this issue); and a University of Chicago group has developed a microfluidic-based consumable chip for several life sciences applications, including digital PCR, and has optimized it for use with several isothermal amplification methods, including LAMP (PCR Insider, 10/7/10).
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