HRM for the Detection of Important Poxviruses

A high resolution melt (HRM) is a powerful, post-PCR, analysis technique used to identify the DNA products present.

First of all PCR is performed with an intercalating dye present. These dyes have the unique property of only binding to double-stranded DNA. Once incorporated into the newly synthesised amplicon the dye will fluoresce brightly.

Performing a high-resolution melt exposes the DNA to a rise in temperature, typically from 50°C to 95°C. When the amplicon reaches its specific melting temperature its DNA denatures and becomes single-stranded. Longer fragments and/or those with a higher GC content will have higher melting temperatures. As the DNA melts and becomes single-stranded, the intercalating dye dissociates from the DNA, and the level of fluorescence decreases. This is used to generate a melt curve that is specific and sensitive enough to detect sequence variations as small as a single-base change.

This principle forms the basis of a new HRM assay “for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance”. Published in nature, the article details a novel multiplex PCR method that utilises genus specific primer pairs and a dsDNA binding dye to generate the PCR amplicons. High-resolution melting curve analysis is then used to discriminate the products of varying fragment sizes and GC contents.

 

Transmission electron micrograph of a smallpox virus particle

(Dr Graham Beards at en.wikipedia)


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