application of real-time its-1 restriction fragment melting curve analysis to detect leishmania species: a gel free and rapid approach

Leishmaniasis, a disease endemic in the middle east region of asia, is primarily carried by dogs. conventional methods like microscopical evaluation and culture have limited the detection of leishmania species. the hae iii rflp pcr method, based on the internal-transcribed-spacer-1 (its-1) region, has been proposed for detecting leishmania species. however, this method is time-consuming and requires agarose gel electrophoresis. a study was conducted on skin samples from naturally infected dogs at shiraz university s clinic. twenty samples were collected and subjected to dna extraction using a commercial kit. the pcr reactions targeted the ribosomal its1 region usingprimers(lsgits1f1:5’cattttccgatgattacac3’,lsgits1r1:5 cgttatgtgagccgttatc-3).to identify leishmania species, the pcr products were digested using haeiii restriction enzymes. the melting curve of different its-1 restriction fragments of different species was analyzed using sybergreen qpcr mastermix and a real-time device. samples from each melting curve analysis and pcr-rflp profiles were sent for direct sequencing and compared with other sequences in the ncbi database using blast. comparison of the results showed that real-time its-1 restriction fragment melting curve analysis provided good accuracy and precision for identification of leishmania species. the normalized melting curves of each three species of leishmania represented different trends and colors, with l. tropica, l. major, and l. infantum exhibiting red, orange, and blue colors and variations in melting temperatures. this method can replace gel electrophoresis in rflp-pcr as a faster diagnostic method.