Identification and Differentiation of Iranian Leishmania Species by PCR Amplification of kDNA

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Abstract
We describe the specific identification of Leishmania species in Iran using PCR DNA amplification of kDNA. For this purpose, we designed a pair of primers - upstream 5' TCGCAGAACGCCCCTACC 3' and downstream 5'-AGGGGTTGGTGTAAAATAGGC 3' - specific for conserved sequences of kDNA of Leishmania. Using this primer, we identified 3 different amplified fragments from the kDNA of the WHO reference Leishmania species. Two bands at 620 and 850 bp were identified for L. major (MRHO/IR/64/Nadim-1 strain) and only 1 band at 620 bp was identified for L. major (P strain). Therefore, we could differentiate 2 Leishmania species. Also, 1 band at 830 bp was identified for L. tropica (MHOM/Sudan/58/OD strain). We determined the sequence analysis of 2 DNA bands (620 and 850 bp) obtained from kDNA of L. major (MRHO/IR/64/Nadim-1). A total of 157 bp from the 5' site and 234 bp from the 3' site were sequenced and showed about 28% homology between 620 and 850 bp fragments. This technique could amplify as little as 1 fg of DNA and was used to differentiate kDNA samples isolated from Iranian patients with cutaneous leishmaniasis. These data indicate that the primer used for PCR amplification of kDNA is specific and can be used for diagnostic and epidemiological purposes.