Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

Abstract

Background: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification.Methods: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High-Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis.Results: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol.Conclusions: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

HRM Accuracy and Limitations as a Species Typing Tool for Leishmania Parasites

“…There are recent studies in the literature addressing the use of HRM to identify Leishmania species [17,31,32]. None, however, have tested the protocol accuracy in a significantly bona fide strains panel representing the inter-and intraspecific diversity of Leishmania.…”

Rapid detection, quantification and speciation ofLeishmaniausing real-time PCR and DNA sequencing at the rRNA Internal Transcribed Spacer 2

“…More recently diagnosis has been facilitated by advances in molecular biology and the PCR amplification and sequencing of various genes has allowed for significantly faster speciation of Leishmania parasites directly from clinical samples. A range of genes have been utilized for speciation including 7SL, HSP70, and CytB, amongst others (11)(12)(13)(14)(15)(16).…”