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


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.


CONCLUSIONS
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.
Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
S
S
Khademvatan
N
N
Neisi
S
S
Maraghi
J
J
Saki
D000818:Animals; D001399:Azure Stains; D016054:DNA, Protozoan / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D007891:Leishmania / Q000145:classification* / Q000302:isolation & purification; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis* / Q000469:parasitology; D008853:Microscopy; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D001399:Azure Stains; D016054:DNA, Protozoan