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Pathogen detection by massively parallel HRM analysis

Bacterial infection identification often still relies on time-consuming culture-based and phenotypic identification methods; however, PCR-based systems are making huge advances in detection speed and sensitivity. High resolution melt (HRM) has become a popular and inexpensive technique for DNA sequence characterization. By accurately measuring the fluorescence during the heating of post-PCR DNA amplicons in the presence of a fluorescent intercalating dye (such as EvaGreen® dye), sequence-specific DNA melting curves are generated.

In a recent article published in Scientific Reports, authors Velez et al. describe a bacterial pathogen detection approach called universal digital high resolution melt (U-dHRM). Their integrated system contains a microfluidic chip and instrumentation to perform PCR amplification with universal bacterial 16S gene primers, high resolution melting, and simultaneous detection within 20,000 picoliter scale reactions. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The authors demonstrated that at least 37 clinically relevant organisms could be distinguished by U-dHRM.

The high specificity and dynamic range of this platform, and its ability to simultaneously identify multiple pathogens, could make a positive impact in infectious disease diagnostics.

To read the original article, click here. Using HRM may require a license from Idaho Technology or Roche Applied Science.

Velez, D. O., et al. (2017). Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling. Scientific Reports, 7, Article number: 42326. doi:10.1038/srep42326

EvaGreen® dye binds to dsDNA via a novel “release-on-demand” mechanism.

EvaGreen® dye is the only qPCR dye that can be used for both real-time PCR and high resolution melt curve analysis. It also has applications in isothermal DNA amplification, droplet digital PCR and more. In addition, EvaGreen® dye is non-toxic, non-mutagenic, and not hazardous to aquatic life. To learn more about EvaGreen®, click here.