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Droplet Size Used as qPCR Readout in Prototype Device to Detect Heart Infections


NEW YORK (GenomeWeb) – Researchers at the University of Arizona have developed a droplet-based qPCR device that can detect bacterial nucleic acids directly from tissue samples in less than four minutes.

In a proof-of-principle study published last week in Science Advances, Jeong-Yeol Yoon and colleagues showed that the device could be used to detect genes conferring resistance to the antibiotic vancomycin from bacteria that were spiked into porcine heart tissue samples, a model of infective endocarditis.

The method can yield quantitative results in fewer cycles than traditional fluorescence-based qPCR, with times as fast as 28 seconds per cycle and subpicogram limits of detection.

Streptococci, enterococci, and Staphylococcus aureus are the most common causes of infective endocarditis. Vancomycin is the last resort for treating methicilllin-resistant S. aureus, or MRSA, but strains of vancomycin-resistant S. aureus (VRSA), as well as vancomycin-resistant enterococci (VRE), have been discovered in clinical cases.

The Science Advances study measured the 16S rRNA hypervariable regions V1-V2 and the vanA gene from vancomycin-resistant Enterococcus faecium.

Diagnosing endocarditis by PCR would be quicker than culture, but tissue tends to inhibit PCR if used directly.

The new method — called droplet-on-thermocouple silhouette real-time PCR, or DOTS qPCR — overcomes PCR inhibition, and relies on the observation that ongoing nucleic acid amplification in a droplet containing an intercalating dye can cause the droplet to shrink.

The inhibition reduction is due to so-called "interfacial effects," and has the added benefit of doing away with fluorescence detection.

"We actually found this phenomenon by accident," Yoon, an engineering professor at the University of Arizona, told GenomeWeb.

In droplets, tissue contaminants are relatively hydrophobic and of high molecular weight so they adsorb to the water-oil interface, effectively removing them from the aqueous droplet, Yoon explained.

"That's how we achieve the inhibition relief," he said.

In fact, the method works best with PCR contaminants present, Yoon said, because the contaminants saturate the water-oil interface and prevent the dNTPs, primers, and other PCR components with hydrophobic tendencies from adsorbing.

The group had initially used a compound referred to as DSA as a "passivating" molecule, essentially creating a shell at the interface to lock the PCR reaction inside the droplet, Yoon said.

But, by playing with variables, the group found that tissue contaminants seemed to serve the same purpose.

Yoon's team also initially fabricated a smartphone-based imaging device in order to monitor fluorescence change in droplets during nucleic acid amplification.

The problem was that as soon as the researchers added the intercalating dye, the droplet began shrinking, totally disappearing after 15 to 20 thermal cycles. 

"We found that this was consistent, and we did more experiments to find that interfacial tension was decreasing, meaning that we were losing droplet over time."

Yoon explained that when the SYBR Green intercalating dye binds to double-stranded DNA, it neutralizes the DNA molecule, making it relatively more hydrophobic.

The neutrality and high molecular weight of freshly-amplified products drives them to the water-oil interface, eventually allowing them to escape into the oil, diminishing the droplet's volume.

The word "silhouette" in the name of the method is to indicate that the device is monitoring the shape of the droplet, using this as a real-time quantification tool, Yoon explained.

"That is our mechanism of doing detection," he said. "It has never been demonstrated before, and it is much faster than conventional fluorescence detection."

In the device, the thermocouple loop that the droplet sits in provides feedback on droplet temperature. When the target temperature is reached, it triggers an arm holding the droplet to zip to another location in the device for the next thermal step.

Noticeable fluorescence change does not happen until 20 to 25 cycles in typical qPCR, Yoon said. But the shrinking phenomenon was robust enough that it enabled visual detection of PCR amplification after three to eight thermal cycles.

It might now soon be possible for doctors to detect infectious agents in the operating room during valve replacement surgery, rather than running conventional qPCR during the operation. Furthermore, the total cost of all components to build the DOTS qPCR device is less than $20, according to the study, and it is disposable.

Yoon is collaborating with other University of Arizona labs to further develop the technology, and he said he has already been contacted by two interested companies. But he hopes the new publication will also attract other industry collaborators, particularly to help with clinical trials.

As previously covered, the researchers have been developing the DOTS qPCR technology as a potential Ebola virus assay, a project that Yoon said is ongoing. He also expects to publish a simplified version of the detection platform within a year.