Volume 21, Issue 1 (Paramedical Sciences and Military Health- Spring- 2026)
Paramedical Sciences and Military Health 2026, 21(1): 4-13 |
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Jalallou N, Zamani S A, Ghorbani M, Vahidi M, Shakouri M. Design and Development of a Multiplex PCR Method for Identification of Leishmania tropica and Leishmania major. Paramedical Sciences and Military Health 2026; 21 (1) :4-13
URL: http://jps.ajaums.ac.ir/article-1-544-en.html
URL: http://jps.ajaums.ac.ir/article-1-544-en.html
1- Department of Laboratory Sciences, School of Allied Medical Sciences, AJA University of Medical Sciences, Tehran, Iran. & Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran. , Nahidjalallou@yahoo.com
2- Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran.
3- Department of Laboratory Sciences, School of Allied Medical Sciences, AJA University of Medical Sciences, Tehran, Iran. & Research Center for Cancer Screening and Epidemiology, AJA University of Medical Sciences, Tehran, Iran.
2- Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran.
3- Department of Laboratory Sciences, School of Allied Medical Sciences, AJA University of Medical Sciences, Tehran, Iran. & Research Center for Cancer Screening and Epidemiology, AJA University of Medical Sciences, Tehran, Iran.
Abstract: (19 Views)
Introduction: Cutaneous leishmaniasis is a major public health problem in Iran that mainly caused by two species: Leishmania major (rural/moist type) and Leishmania tropica (urban/dry type). Accurate and rapid differentiation of these two species is essential for choosing appropriate treatment and controlling the disease. Traditional microscopic methods have low sensitivity and they cannot distinguish species with identical morphology, whereas molecular methods are considered more sensitive and specific tools. This study aimed to design and optimize a Multiplex PCR method for the simultaneous molecular identification of these parasites using specific genetic targets including ITS2, COII, and AAP3 from the two species.
Materials and Methods: Genomic DNA was extracted from standard strains of the two species using the phenol-chloroform method and its quality was confirmed by Nanodrop spectrophotometry and gel electrophoresis. Specific primers for the aforementioned genes were designed based on sequences available in the NCBI GenBank and verified by BLAST analysis. The *GAPDH* gene (110 bp) was used as an internal control. Target fragments were initially amplified by single PCR and then cloned into the pTZ57R/T vector. Recombinant plasmids were transformed into *E. coli* Top10 competent cells, confirmed by colony-PCR, extracted, and used as positive controls. Subsequently, duplex PCR (multiplex) reactions were optimized separately for each species and the assay sensitivity was determined by preparing serial ten-fold dilutions of plasmids and calculating the limit of detection (LOD) based on copy number.
Results: The AAP3 (468 bp) and COII (213 bp) gene fragments for L. major , and ITS2 (200 bp) and COII (442 bp) for L. tropica , were successfully amplified from genomic DNA extracted from standard strains. The band sizes matched software predictions. The LOD for L. major was 19 copies for AAP3 and 41 copies for COII; while, for L. tropica, it was 285 copies for ITS2 and 44 copies for COII. The optimized multiplex reactions clearly resolved distinct bands corresponding to both target genes alongside the internal control with appropriate resolution.
Conclusion: The designed Multiplex PCR method, employing a combination of nuclear (ITS2), mitochondrial (COII), and functional (AAP3) gene targets, provides a sensitive, specific, and rapid approach for the differential diagnosis of L.major and L. tropica. The recombinant plasmids generated in this study offer safe and stable positive controls replacing live parasite cultures. This approach represents a practical step toward the development of indigenous molecular diagnostic kits for cutaneous leishmaniasis in endemic regions of Iran.
Materials and Methods: Genomic DNA was extracted from standard strains of the two species using the phenol-chloroform method and its quality was confirmed by Nanodrop spectrophotometry and gel electrophoresis. Specific primers for the aforementioned genes were designed based on sequences available in the NCBI GenBank and verified by BLAST analysis. The *GAPDH* gene (110 bp) was used as an internal control. Target fragments were initially amplified by single PCR and then cloned into the pTZ57R/T vector. Recombinant plasmids were transformed into *E. coli* Top10 competent cells, confirmed by colony-PCR, extracted, and used as positive controls. Subsequently, duplex PCR (multiplex) reactions were optimized separately for each species and the assay sensitivity was determined by preparing serial ten-fold dilutions of plasmids and calculating the limit of detection (LOD) based on copy number.
Results: The AAP3 (468 bp) and COII (213 bp) gene fragments for L. major , and ITS2 (200 bp) and COII (442 bp) for L. tropica , were successfully amplified from genomic DNA extracted from standard strains. The band sizes matched software predictions. The LOD for L. major was 19 copies for AAP3 and 41 copies for COII; while, for L. tropica, it was 285 copies for ITS2 and 44 copies for COII. The optimized multiplex reactions clearly resolved distinct bands corresponding to both target genes alongside the internal control with appropriate resolution.
Conclusion: The designed Multiplex PCR method, employing a combination of nuclear (ITS2), mitochondrial (COII), and functional (AAP3) gene targets, provides a sensitive, specific, and rapid approach for the differential diagnosis of L.major and L. tropica. The recombinant plasmids generated in this study offer safe and stable positive controls replacing live parasite cultures. This approach represents a practical step toward the development of indigenous molecular diagnostic kits for cutaneous leishmaniasis in endemic regions of Iran.
Keywords: Leishmania major, Leishmania tropica, Multiplex PCR, Molecular diagnosis, Cutaneous leishmaniasis
Type of Study: Research |
Subject:
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Received: 2026/07/27 | Accepted: 2026/08/8 | Published: 2026/03/10
Received: 2026/07/27 | Accepted: 2026/08/8 | Published: 2026/03/10
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