Researchers have developed a new system that can detect viruses in sample amounts one million times smaller than current commercial instruments and with about half of the analysis steps.This advance, achieved by a team of Lawrence Livermore National Laboratory (LLNL) and University of California (UC), Davis scientists and engineers, will lead to more rapid detection of viruses and other pathogens.
"The advantage of reducing the volume size of the sample is that the fluorescent signal can be seen sooner than in commercial devices because it becomes so much brighter," said Bill Colston, one of the paper's authors and the leader of LLNL's Chemical and Biological Countermeasures Division.
The team's microfluidic system permits polymerase chain reaction (PCR) analysis - or DNA copying - to be performed inside 10-picoliter droplets (about 10 trillionths of a liter) on a silicon chip. It would take about 25 billion of these droplets to fill a cup of water.
Using their PCR-on-a-chip system, the LLNL-UC Davis team has analyzed hundreds of droplets in tests and demonstrated that their core technology works. Now their challenge is to modify this system into one that can process hundreds of thousands and, in time, millions of droplets.
In addition to Colston, other authors of the Analytical Chemistry article are: mechanical engineers Reg Beer and Klint Rose, chemist Ben Hindson, biologist Sara Hall, chemical engineer Elizabeth Wheeler and UC Davis professor of mechanical engineering Ian Kennedy.
The paper's lead author, Beer received his Ph.D. from UC Davis as a part of the project and also designed and developed the microfluidics system.
The new technique significantly reduces the number of PCR heating and cooling cycles required for detecting a pathogen from 40 to about 20.
Early-stage research detailed in the paper, according to Colston, answered a number of important questions, such as whether a sample could be broken into droplets in a controlled manner and whether a single copy of a virus could be detected in a droplet.
The research is part of a larger, LLNL-funded effort - called the Viral Discovery Platform (VDP) - to identify emerging, engineered or unknown viral threats in days rather than weeks or months.
Current bioterrorist agent detection technologies, such as the BioWatch system and LLNL's Autonomous Pathogen Detection System (APDS), can detect the airborne release of a list of known biological agents. However, these systems are not designed to identify or detect an unknown or newly emerging virus.
For example, after the SARS outbreak in 2003, samples of the virus were sent to 20 different laboratories for analysis and it took about 60 days to identify the pathogen as a corona virus, Colston noted.
"We and other researchers see the need to explore ways to determine if the nation can be better prepared for emerging, engineered or other unknown viruses," Colston said.
Currently, researchers who are studying a particular virus take a sample, such as from a nasal swab, and try to isolate and make copies of that virus for their investigations.
With the new LLNL-UC Davis approach, the team hopes to be able to analyze the entire sample, finding the virus in question and also learning about any other viruses in the sample.
"Our goal is take a sample that contains lots of viruses, break it down into small droplets, each of which contains no more than a single virus, and then analyze all of the droplets that have viruses individually," Colston explained.
Early data show that if researchers had hundreds of unique DNA signatures for different viruses, it would be possible to identify all known viruses and to also identify a significant fraction of the emerging viruses, according to Colston.
"We're pleased with our initial results, but we still have some tough technical challenges to overcome," Colson said.
To date, the team has received one patent for its research, with four additional patents pending.
For the future, in addition to seeking to identify all of the viruses present in millions of droplets, the team also would like to develop assays that detect newly emerging or unknown viruses.