Evaluation of DNA melting analysis as a tool for species identification

Abstract

Summary 1. One of the ongoing challenges in molecular biology is to develop rapid, accurate and reliable techniques to identify organisms. This contribution evaluates the value of DNA melt peak analysis for the identification of pests and pathogens significant to biosecurity. 2. The method evaluated in this study capitalises on the observation that double‐stranded DNA separates into single strands (melts) at a temperature dependant on the nucleotide sequence of the DNA strands. The temperature and shape of melt peaks from polymerase chain reaction (PCR) products using standard primers and amplification protocols were evaluated for species identification, and the advantages of this method over agarose gel electrophoresis exemplified. 3. Three insect (weevil) species were discriminated by the melt profile shape and temperature of their cytochrome oxidase subunit I (COI) PCR product using order‐specific universal primers. 4. Three of four arachnid (tick) species were discriminated by the melt profile shape and temperature of their COI and 18S PCR product using order‐specific universal primers. 5. Plum pox virus‐specific primers amplified only the target virus that was verified by its unique melt temperature rather than by conventional staining of the amplicon on an electrophoretic gel. 6. Melt peak analysis is a useful and robust tool for the rapid detection of PCR products and the identification of organisms. An efficiency study revealed this technique required half the time input, and cost half as much, as PCR followed by AGE.