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Gates Foundation, Grand Challenges Grants Highlight New Amplification, Sample Prep Methods

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By Ben Butkus

The Bill and Melinda Gates Foundation and Grand Challenges Canada last week announced $31.5 million in new funding to support the development of point-of-care diagnostic technologies for the developing world.

Although the grants will support technology development in several areas, a significant number of the awards will focus on new innovations in the area of PCR, nucleic acid amplification, and sample prep.

According to the organizations, the grants cover five research areas intended to break down POC diagnostics into component parts: Drawing blood or collecting other biological samples and preparing it for analysis; analyzing the sample to identify disease; obtaining and transmitting data and receiving results; and ensuring the device will work in the field where there is often no electricity or refrigeration.

"The project is analogous to software developers creating new apps for smart phones and tablet computers," Rebecca Lackman, program officer for diagnostics for Grand Challenges Canada, said in a statement. "Researchers have accepted the challenge to create novel sampling and testing systems that can be plugged into a standardized analyzer that can test for malaria, tuberculosis, HIV, and a variety of tropical diseases. The 'Integrated Innovation' approach means they will also investigate the social and business innovations needed for successful product delivery and use."

Lackman also noted that the organizations intend to create a common application platform, thus reducing commercialization costs and regulatory issues, and potentially providing additional incentive for industrial partners to become involved.

The two organizations funded 22 new grants in total, with the Gates Foundation taking the lead on 12 awards and Grand Challenges administering 10. The organizations did not disclose individual award amounts, but a spokesperson from Grand Challenges Canada told PCR Insider that the amount of each grant varies from about $470,000 to $4 million.

A complete list of awardees can be found here. Following is a summary of several projects of note related to PCR, nucleic acid amplification, and sample prep:

Mesa Tech International, a recent spinout of Los Alamos National Laboratory, will develop a disposable sub-system for nucleic acid sample prep that does not require instrumentation or user intervention.

As part of the project, Mesa Tech researchers led by Robert (Bruce) Cary will more specifically develop nucleic acid purification systems that use a novel configuration of lateral flow materials to bind and wash nucleic acids to yield amplification-ready samples. The devices could provide purified samples from clinical specimens within minutes without user intervention, instrumentation, electricity, or costly materials, the researchers wrote in their grant abstract.

In August, Mesa Tech was awarded a $300,000 Phase I Small Business Innovation Research grant from the National Institutes of Health to develop similar technology for multiplexed diagnosis of respiratory diseases (PCR Insider, 8/18/2011).

Luke Lee, a researcher from the University of California, Berkeley, is developing an integrated microfluidic "universal sample prep and pre-concentration," or USP, module, which will enable parallel diagnostics of infectious diseases such as HIV, TB, and malaria.

According to the grant abstract, the module will use electrical and physical methods that will be compatible with different sample inputs and downstream analytical techniques to provide both plasma and cellular biomarkers. The device will not require external reagents, will have low power consumption, and can be operated on site with minimal training.

"Our role is to try and standardize blood sample prep," Lee told PCR Insider this week. "We propose to standardize blood sample prep on a chip about the size of a credit card. We don't have to centrifuge. We are trying to cut off the umbilical cord of any external tubing, so we can basically prick a finger and blood will be processed."

Lee said that his group has already developed one chip that can separate plasma from whole blood. He said that the group will not aim to purify nucleic acid or protein biomarkers, per se. "Why do you need to purify [DNA and proteins] if you can directly detect it? We can selectively lyse white blood cells, or red blood cells, depending on the disease," he said.

Lee added that his group is also developing methods to electrochemically or optically detect a signal from the amplification of nucleic acids.

Ohio State University researchers, led by Besik Kankia, are developing a quadruplex-based technology for isothermal DNA amplification and non-enzymatic detection.

The method will feature a simple fluorescence detection scheme which, if successful, will generate a signal that can be detected by a portable fluorimeter or by eye after excitation with an appropriate light source.

"We have discovered a way to use the potential energy of DNA quadruplexes to perform very simple and efficient DNA amplification, as well as non-enzymatic DNA signal amplification," Kankia further explained in an e-mail to PCR Insider.

"The key point of quadruplex-driven reactions is that some G-rich sequences are capable of forming quadruplexes with significantly more favorable thermodynamics than the corresponding DNA duplexes," Kankia added. These sequences are incorporated within DNA duplexes which, after interacting with an initiator such as pathogen DNA or DNA polymerase, self-dissociate from the complementary strand and fold into quadruplexes.

"The energy of quadruplex formation is used to drive DNA amplification at a constant temperature or non-enzymatic DNA signal amplification," Kankia said.

Cornell University's Daniel Luo and colleagues' project will focus on creating self-amplifying DNA polymers for POC diagnosis using a portable CMOS sensor. The system will use branched DNA and/or light to drive signal amplification.

More specifically, the group will design "specific branched DNA that can fish out pathogen DNA or RNA and then glue them together into giant conglomerates, which can be easily detected by a variety of methods," Luo told PCR Insider in an e-mail.

"This amplification step is totally equipment-free and enzyme-free and only occurs in the presence of specific pathogens," Luo added.

In addition, Luo's group will collaborate with the laboratory of Cornell researcher Edwin Kan to help improve the robustness of the self-amplifying DNA polymer for real-world settings, particularly in the developing world.

For example, the groups have recently demonstrated that the DNA amplification scheme works well, "even in dirty water and a wide range of pH," and in a matter of minutes, Luo said. "The next step is to improve the sensitivity and specificity of the detection," he added. A further goal, he noted, is to develop a miniaturized and integrated detector that is based on CMOS and will allow results to be further integrated with cell phones and GPS systems.

Other notable projects include:

• Bigtec Labs of Bangalore, India, will create a low-cost automated sample preparation system that can be interfaced with nucleic acid detection techniques and will involve the creation of a device to extract pathogenic DNA/RNA from biological samples such as blood, sputum, urine, and nasal/throat swabs.

• David Beebe and colleagues at the University of Wisconsin, Madison, will develop a microfluidic immiscible phase barrier to enable simplified sample preparation for POC diagnostics in the developing world. The platform will be readily adaptable to various upstream collection components, and the immiscible phase barrier will produce a clean sample for output to downstream amplification and detection components.

• A group at the California Institute of Technology, led by Axel Scherer, a co-founder of recent Illumina acquisition Helixis, has received a grant for a project entitled "Quantitative PCR Reconceived for Developing World Point-of-Care Diagnostics." Along with collaborators at Dartmouth University, Scherer and colleagues will develop a prototype qPCR amplification and detection component module that can rapidly detect a wide range of pathogens with low cost, low internal and outward complexity, low power consumption, a small size, and a rugged design, according to the grant's abstract.

• Scientists at Usar Biotechnologies in Hangzhou, China, will develop an equipment-free nucleic acid extraction system and isothermal amplification platform under a Grand Challenges grant. The proposed system will be affordable, rapid, and simple enough to be performed at the village level by minimally trained personnel, according to the grant's abstract.


Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.

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