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European Researchers Enable Single-Dye Multiplex qPCR via Melt-Curve Analysis in Each Thermal Cycle

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NEW YORK (GenomeWeb) – Multiplex qPCR typically requires a separate fluorophore and optical channel for each target in a sample, but a proof-of-principle methods paper has now described a way to multiplex using only a single dye by embedding melt analysis within the PCR thermal cycling itself.

The technique, published last week in Scientific Reports, uses EvaGreen DNA intercalating dye and dynamically extracts fluorescence and temperature data during the transition between extension and denaturation in each PCR cycle, Pavel Neužil, an author on the paper, told GenomeWeb in an email.

Neužil, who is affiliated with the European branch of the Korea Institute of Science and Technology in Saabruecken, Germany called KIST-Europe as well as Brno University of Technology in the Czech Republic, said the method has two main requirements. There must be a minimized temperature gradient during the transition and at least 50 measurements during that period of time.

The study used a special "virtual reaction chamber" consisting of a 150 nanoliter droplet of sample encapsulated with an oil droplet and placed on hydrophobically coated glass.

"Underneath the glass is a micromachined silicon heater, [and] this heater also serves as a heat sink due to the high thermal conductivity of silicon," Neužil explained.

The study noted that the small thermal mass of this setup and the special heater led to greater than 20 degrees Celsius per second heating and cooling.

"The sample warms up from an annealing temperature of 72 degrees Celsius to a denaturing temperature of 93 degrees Celsius very fast, in one to two seconds, and the fluorescent detector has to be able to conduct at least 50 measurements during that period of time," Neužil noted.

The Scientific Reports study applied the method to the detection of genes from the H7N9 influenza strain. This bird flu virus is usually detected by measuring the hemagglutinin and neuramidase genes, as well as their ratio, and multiplex PCR is the method of choice. The study detected these genes from cDNA spiked into master mix and suggested that a difference in amplicon melting temperature of at least five degrees is sufficient to differentiate two targets.

Since melt temperature is a function of amplicon length and CG/AT ratio, primer design could be used to tweak the melts for other amplicons. An extension temperature of 60 degrees Celsius and denaturation temperature of 95 degrees Celsius would theoretically allow for six different amplicons to be detected in the same droplet, the study concluded.

The researchers have also built a handheld device to enable this process, and that will be the subject of a forthcoming paper.

"The commercial PCR we had available … could not perform enough measurements during the transition period," Neužil noted, adding that there may be faster systems, but his group did not have access to them at this time.

The researchers are now considering the best way to commercialize the handheld PCR device.

"One thought is to start with the education market to supply educational institutions with this molecular diagnostic tool, and at the same time conduct clinical trials [for potential use] in the point-of-care diagnostic market," Neužil said.

The researchers are also "on the lookout for commercialization partners and funding," Neužil said. The handheld PCR prototype has been tested in collaboration with a researcher in the Nanofabrication Research Group at the National Institute of Standards and Technology in the US, and the team is finishing a manuscript now.

"We believe that we have developed the smallest real-time qPCR [device] in the world, as it can be held in one hand, and is capable of testing four samples at once in as fast as six minutes," Neužil said.

This handheld device is standalone and does not require any accessories, he said. It has an integrated fluorescent system, a heater, all control electronics, as well as a small LCD display, and will show temperature profiles during PCR progress propagation and the final PCR amplification curves. The device requires only a 12 volt power supply, Neužil said.

"We also have a development pipeline involving a few generations of digital PCR prototypes testing between 1 and 2 million samples at a time, with a system cost of about only $100," he said.

Neužil said he thinks the final packaging of the qPCR system should be redesigned by an industrial product engineer, but he expects the cost of manufacturing to be about $200 and the cost per run to be only a few cents — essentially just the cost of nanoliter-sized droplets of PCR master mix and EvaGreen dye.

Of note, a somewhat similar method — using a single dye for multiplex telomere length detection — was published in Nucleic Acids Research in 2009, and a patent application for "monochrome multiplex quantitative PCR" was filed in the US around that time. But that method differs in that it quantifies amplicons via an additional step.

"We can detect all amplicons simultaneously … as we do it dynamically," Neužil said.

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