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1.  Cytochrome b and Molecular Typing of Leishmania spp. in a Passive Sampling of Suspected Patients with Cutaneous Leishmaniasis in Sistan and Baluchestan Province, Eastern Iran 
Iranian Journal of Parasitology  2017;12(4):534-543.
Despite the high prevalence and drug resistance of disease in Sistan and Baluchestan Province of Iran, the species of cutaneous leishmaniasis (CL) has not been identified. In the present study, cytochrome b (Cyt b) was used in Sistan and Baluchestan to find species of Leishmania in suspected patients of CL using PCR-RFLP and DNA sequencing.
This study was conducted from Oct 2015 to Oct 2016. The samples were collected from the individuals clinically suspected to CL and referred to Iran Shahr, Chabahar, Khash, Zabol, Zahedan, Mirjaveh, and Nikshahr health centers. Overall, 700 Giemsa-Stained slides from the wound of patients suspected of CL were passive collected and examined under a light microscope at ×1000. After DNA extraction, positive samples were used for Cyt b detection by PCR-RFLP to determine the parasite species. One hundred positive slides were selected for molecular studies. Among positive samples, 20% were sequenced. To compare the results of sequences, molecular evolutionary genetic analysis (MEGA6) was used.
Overall, 53 samples were identified as L. major and 47 samples (47%) L. tropica. Cyt b in L. major and L. tropica is converted to 400 and 480 bp and 130, 215 and 535 bp pieces respectively. In the isolated L. tropica and L. major, nucleotide changes were 3–5 (mainly in wobble site).
Infection was more related to L. major. PCR-RFLP method has a high sensitivity for diagnosis of Leishmania species.
PMCID: PMC5756303
Leishmania major; Leishmania tropica; Cytochrome b; PCR; DNA sequencing; Iran
2.  Species Identification and Molecular Typing of Leishmania Spp. Using Targeting HSP70 Gene in Suspected Patients of Cutaneous Leishmaniasis from Sistan and Baluchestan Province, Southeast Iran 
Iranian Journal of Parasitology  2016;11(4):489-498.
Leishmaniasis is a sand fly-borne disease caused by the protozoan parasites belonging to the genus Leishmania. Because of the preventing and controlling methods, clinical course, prognosis and choice of treatment are differing from species; differentiation of species is critical. The present study was aimed to detect the parasite species using the PCR-RFLP method.
A total of 130 Giemsa-Stained slides from suspected Cutaneous leishmaniasis (CL) patients were examined under a light microscope at ×1000. DNA from each slide was extracted PCR method was undertaken with HSP70 genes and the PCR products were digested with a restriction enzyme HaeIII (BsuR1). The study was conducted in the laboratory of Zahedan University of Medical Sciences in the Sistan and Baluchestan Province, southeastern Iran in 2015.
From 130 suspected samples, 59 (45.3%) were positive by the microscopic examination, meanwhile 64 (49.2%) were positive by PCR-RFLP, Leishmania species were recognized, and L. tropica was introduced as predominant species in current study.
PCR-RFLP is a valuable technique for distinguish of Leishmania species. Furthermore, anthroponotic CL is the dominant cause of CL in Sistan and Baluchestan Province.
PMCID: PMC5251177  PMID: 28127360
Leishmania; HSP70 gene; PCR-RFLP; Human; Iran
3.  Cloning, expression and immunoreactivity of recombinant Toxoplasma gondii GRA5 protein 
Iranian Journal of Microbiology  2016;8(5):331-337.
Background and Objectives:
Toxoplasma gondii is an obligatory intracellular parasite which causes severe diseases in the fetus of pregnant women and immunocopmromised patients. Serological tests based on recombinant protein are one of the main diagnosis methods for the detection of specific antibodies in serum samples. Dense granule antigenic proteins derived from T. gondii (TgGRAs) are potential antigens for the development of diagnostic tools.
Materials and Methods:
DNA was extracted from T. gondii (RH-strain) tachyzoites and PCR reaction was done using corresponding primers for GRA5 antigen. The PCR product was purified and ligated into pTG19-t vector and then subcloned into XhoI and BamHI digested pGEX6p-1 expression vector. Recombinant plasmid was transformed into E. coli (BL21 DE3) and induced by 1mM IPTG and analyzed by 15% SDS-PAGE. Expressed protein was confirmed by western blot analysis.
There was no difference among the sequences of T. gondii GRA5 gene from different isolates. The recombinant plasmid pGEX-6p-1/GRA5 induced by IPTG was expressed in E. coli. It was a GST fusion protein and could react with human positive sera analyzed by western blot.
The GRA5 gene of T. gondii isolates is highly conservative. This antigen as a recombinant protein was successfully expressed in E. coli, which showed high immunoreactivity.
PMCID: PMC5277603  PMID: 28149494
Toxoplasma gondii; Dense granule antigen; GRA5; Immunoreactivity
4.  Cloning and Sequence Analysis of Recombinant Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-142 kDa) In pTZ57R/T Vector 
Iranian Journal of Parasitology  2015;10(2):197-205.
Carboxy-terminal 42 kDa region of Plasmodium vivax merozoite surface protein-1 is considered as an important antigen in blood stage. Since, this region has been observed to be polymorphic among isolates of P. vivax, it is significant to survey on different regions of this antigen in various areas of the world.
In the present study, the genetic diversity of cloned PvMSP-142 kDa gene from an Iranian patient is analyzed. Parasite DNA was extracted from a P. vivax - infected patient in Iran. The region of PvMSP-142 kDa was amplified by PCR, cloned into pTZ57R/T vector and then sequenced.
Sequencing of cloned PvMSP-142 kDa gene clearly has a high degree of homology (95%) with reference Sal-I sequence and also with the homogeneous sequences from some studied countries (97%). Thirty eight SNPs (single nucleotide polymorphism) were identified in cloned PvMSP-142 kDa gene which the mutations had localized in the 33 kDa fragment (PvMSP-133 kDa), while there was nearly no variation in the 19 kDa fragment (PvMSP-119 kDa). 2 out of 38 mutations were found as to be novel haplotypes.
High similarity of cloned PvMSP-142 kDa gene in comparison to reference sequence and other sequences could be beneficial as a remarkable molecular marker for serological diagnostic kits of P. vivax in malarious neighboring countries of Iran and around the world.
PMCID: PMC4522295  PMID: 26246817
Plasmodium vivax; Recombinant MSP-1 42 kDa; Sequencing; Iran
5.  High-Level Expression of Immunogenic Recombinant Plasmodium vivax Merozoite Surface Protein (Pvmsp-142 kDa) in pGEX 6P1 Vector 
Detection of Plasmodium vivax specific antibodies with serological tests could be a valuable tool for epidemiological researches. Whereas P. vivax cannot be simply obtained in vitro, serological tests using total or semi-purified antigens are infrequently used. Given this restriction, the present study investigated whether recombinant P. vivax merozoite surface protein 1 (PvMSP-1 42 kDa) could be useful in detection of antibodies from the serums of a P. vivax infected person using serological tests.
Parasite DNA was extracted from blood sample of an Iranian P. vivax-infected patient. The region of PvMSP-142 kDa was amplified by PCR then cloned into pTZ57R/T vector and sequenced. The insert was sub cloned into pGEX 6P1 expression vector. Afterwards, it was transformed into E. coli BL21 and cultured massively. Sub cloning of gene was confirmed by PCR and enzyme digestion and sequencing finally. Production of recombinant protein was confirmed by SDS-PAGE. Western blot was performed by human sera to appraisal binding ability to the IgG antibodies of P. vivax infected patients. Recombinant protein was purified and estimated by Bradford assay.
The specialty values of the Western blot determined with 10 sera from naturally infected individuals, 10 sera from healthy individuals and 7 sera from individuals with other infectious diseases.
For the Iranian population, using a Western blot assay for MSP-142 recombinant protein can be used as the foundation for promotion of serological assay for the detection of P. vivax malaria such as ELISA.
PMCID: PMC4450018  PMID: 26060780
Plasmodium vivax; Recombinant PvMSP-142 kDa; Expression vector; Iran
6.  Genotyping and Phylogenetic Analysis of Fasciola Spp. Isolated from Sheep and Cattle Using PCR-RFLP in Ardabil Province, Northwestern Iran 
Iranian Journal of Public Health  2014;43(10):1364-1371.
The aim of this study was to detect the genotype of Fasciola spp. in Meshkin-Shahr, Ardabil Province, northwestern Iran in different hosts using PCR-RFLP.
The parasite hosts included cattle, and sheep. Overall, 70 adult flukes from livers of slaughtered animals were collected from the abattoirs of aforementioned area. The included 35 samples from infected sheep and 35 samples from 35 infected cattle. PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer (ITS 1) region from Fasciola species were used to conduct the study.
The fragment of approximately 700bp in all of the Fasciola samples was amplified. PCR products of ITS 1 were subjected for digestion by restriction enzyme. RsaI restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. Amplicons with the sequences of F. hepatica had a pattern of about 360, 100, and 60 bp band size, whereas F. gigantica worms had a profile of 360, 170, and 60 bp in size, respectively. Results based on PCR-RFLP analysis were confirmed by sequence analysis of representative ITS 1 amplicons. No hybrid forms were detected in the present study. All sheep were infected with F. hepatica but cattle were infected with both species.
Both species of Fasciola are present in Ardabil. The method described here can be valuable for identification of Fasciola species in endemic parts for fasciolosis, regions with intermediate species and in that overlapping distribution area.
PMCID: PMC4441889  PMID: 26060698
Fasciola; Genotyping; Fasciolosis; PCR; Iran

Results 1-6 (6)