Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

Abstract

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

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

“…No distinct extensively accepted method is available to identify Leishmania species and strains today. A valuable molecular technique is needed to be developed to separate Leishmania parasites in primary infections 1 , 4 , 7 .…”

“…Leishmaniasis, one of the neglected tropical and subtropical diseases, has been considered based on the medical importance; besides, visceral leishmaniasis is potentially lethal [1][2][3] . From all known Leishmania species, some are medically important.…”

“…The Giemsa staining, clinical indications, vector estimations, in vivo pathogenicity, growth in media, and isoenzyme were used for distinguishing between different Leishmania parasites. Later PCR, RFLP (Restriction Fragment Length Polymorphism), sequencing, and phylogenetic analysis were applied 1 , 7 .…”

Establish an allele-specific real-time PCR for Leishmania species identification

“…There are also some real-time PCR identification methods were developed with different detecting targets, such as cathepsin L-like cysteine protease B gene for L. major , L. tropica and L. aethiopica [ 38 ]; amino acid permease 3 (AAP3) and COII for L. major and L. tropica [ 39 ], and glucose phosphate isomerase (GPI) for Leishmania ( Viannia ) spp . , L. mexicana complex, L. infantum / donovani complex and L. major complex [ 31 ].…”

Development of a Taq-Man qPCR assay for trypanosomatid multi-species detection and quantification in insects

“…For instance, infections with T. cruzi have been determined in infected human blood samples using kDNA and nuclear satellite DNA targets with detection limits of 0.23 and 0.70 parasite equivalents/mL [46]. Leishmania species have been detected with a sensitivity down to 3 copies of parasite per reaction using COII (10-20 copies in each mitochondrion) or the AAP3 (two copies in the parasite genome) genes [47]. Few reports have attempted so far for the multispecies identi cation using TaqMan qPCR.…”