Diagnosis and identification of Leishmania spp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran

• Published in: Annals of tropical medicine and parasitology Vol. 105; no. 8; pp. 559 - 565
• Main Authors: Khademvatan, S, Neisi, N, Maraghi, S, Saki, J
• Format: Journal Article
• Language: English
• Published: Leeds Taylor & Francis 01.12.2011; Maney; Maney Publishing
• Subjects: Animals; Azure Stains; Biological and medical sciences; DNA, Protozoan - analysis; DNA, Protozoan - isolation & purification; General aspects; Humans; Leishmania - classification; Leishmania - isolation & purification; Leishmaniasis, Cutaneous - diagnosis; Leishmaniasis, Cutaneous - parasitology; Medical sciences; Microscopy; Original; Real-Time Polymerase Chain Reaction - methods; Sensitivity and Specificity; Transition Temperature; Iran; Asia; Kinetoplastida; Polymerase chain reaction; Protozoa; Melting; Leishmania; Tropical medicine; Identification; Diagnosis; Molecular biology
• ISSN: 0003-4983; 1364-8594
• DOI: 10.1179/2047773211Y.0000000014

Background: The aim of present study was describing a real-time PCR assay for the diagnosis and direct identification of Leishmania species on Giemsa-stained slides in south-west of Iran. Materials and methods: Altogether, 102 Giemsa-stained slides were collected from different part of south-west of Iran between 2008 and 2011. All the Giemsa-stained slides were examined under light microscope. After DNA extraction, real-time PCR amplification and detection were conducted with fluorescent SYBR Green I. For identification, PCR products were analysed with melting curve analysis. Results: One hundred and two archived slides from suspected lesion examined by microscopy and real-time PCR. The sensitivity of the real-time PCR on Giemsa-stained slid was 98% (96/102). The melting curve analysis (T m ) were 88·3±0·2°C for L. tropica (MHOM/IR/02/Mash10), 86·5±0·2°C for L. major (MHOM/IR/75/ER) and 89·4±0·3°C for L. infantum (MCAN/IR/97/LON 49), respectively. Conclusion: This study is first report in use of real-time PCR for diagnosis and identification of Leishmania spp. in Iran. Up to now, in Iran, the majority of identification of Leishmania species is restriction fragment length polymorphism (PCR-RFLP) of ITS1 and kinetoplast DNA. Our data showed that Giemsa-stained slides that were stored more than 3 years, can be use for Leishmania DNA extraction and amplification by real-time PCR. Compared to conventional PCR-based methods, the real-time PCR is extremely rapid with results and more samples can be processed at one time.