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Researchers Describe Rapid, Multiplexed High-Res Melt Assay for Species Differentiation

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NEW YORK (GenomeWeb) – Differentiating species using real-time PCR is critical to clinical and forensic labs, but developing inexpensive and rapid workflows for high-throughput testing is not always straightforward.

Now, researchers at Towson University in Maryland have described a fast and low-cost method to develop species differentiation assays using high-resolution melt temperature and a saturation dye from BioMérieux subsidiary BioFire Defense. Published in Analytical Biochemistry, the method enables multiplexing with differentiation by melt temperature rather than by color.  

Corresponding author Kelly Elkins is a professor at Towson and teaches in an undergraduate and masters program for forensics science and chemistry accredited by the Forensic Science Education Programs Accreditation Commission. Her team's multiplex molecular assay may have an application in biodefense and foodborne pathogen testing, as it detects and differentiates Salmonella enterica substrain typhimurium, Escherichia coli, and Shigella flexneri. These three bacteria are considered potential bioterror agents by the US Centers for Disease Control and Prevention.

However, it has other potential forensic utility as well, as the authors noted that it can be used to differentiate plant species like marijuana, morning glory, and jimson weed, which will be the subject of a subsequent report on differentiating so-called "legal high" species. That test has also been run with case-work samples provided by Maryland State Police, Elkins said.

The method can also be adapted for clinical and research biochemistry, Elkins said.

The workflow uses BLAST searching for primer design. It then uses a program called OligoCalc to estimate the melting temperature of the PCR products. The program has been around since the late 1990s and is freely available online from Northwestern University.

"Before we went and purchased the primers and tried out the assay in the lab for our multiplex and assay development, we tested right there at OligoCalc to determine if, from the DNA sequence, the length, and the GC content, it would produce an amplicon of a different melt temperature than the amplicons that we already had in the assay," Elkins said.

That computer work saved considerable time and money, and the results were within a few percent of the OligoCalc calculations, she said.

The group also used internal positive controls in the assay that were from commercial kits, and also designed its own IPC.

Initially, the food-borne pathogens assay was developed using SYBRgreen, but that dye "was not giving us the sensitivity with the melts that we needed," Elkins said.

She then read some reports that used a dye called LCGreen Plus from BioFire Defense. "Immediately when it came in, I went to the lab and tried it, and the first test got beautiful results," she said.

LCGreen Plus dye was originally developed for mutation scanning and SNP detection, Matt Scullion, vice president of sales and marketing at BioFire Defense told GenomeWeb. "This article presents an interesting application and demonstrates the excellent performance of the dye," he commented.

The dye was developed when BioFire was still an independent company and called Idaho Technologies. Other so-called saturating dyes, which do not inhibit PCR even if used at concentrations that give maximum fluorescence, were later identified, including EvaGreen and SYTO9.

These all give "much more sensitive melt curves" than other dyes, Elkins explained.

Previous work had shown HRM could be used to differentiate species, and LCGreen Plus has been used for a number of clinical applications. Studies from the lab of Carl Wittwer at the University of Utah in the mid-2000s showed the dye could be used to create multiplex assays to distinguish three mycobacteria species as well as four Aspergillus species using rapid-cycle PCR followed by high-resolution melt curve analysis.

The novelty of Elkins' study was that most other work used melt-curve difference plots and kinetics of the melt curves. "While the data are interesting, it appears that it is easier to automate the interpretation of high-resolution melt assays when you differentiate based on the melt temperature," Elkins said.

Additionally, one previous example differentiated by melt temperature, but only of a duplex assay. Elkins said she had not seen other assays differentiating multiple species in the same assay, specifically using melt temperature in this way.

"The idea of being able to see a triplex or a tetraplex is pretty exciting, and it is pushing the boundaries of HRM," Elkins said.  

The method is easily extensible to other projects and interests. The LCGreen master mix costs about $1 per assay, and the method can be run on standard PCR instruments with HRM capabilities. Elkins' lab runs 72 samples at a time in about two hours, but she said the run time has not been optimized yet and will likely be shortened in the future. And, for bioterrorism, which might require sampling sick individuals and food from many sites, high throughput is a necessity. 

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