title: Design and Validation of Real-Time PCR: Quantitative Diagnosis of Common Leishmania Species in Iran.
abstract: OBJECTIVE
  Design and validation of Real-time PCR on the protected gene region ITS2 to quantify the parasite load in common leishmania (L) species.
  
  
  METHODS
  Probe and primer were designed from the ITS2 region between the rRNA genes with minimum gene variation in three common leishmania species followed by a Real-time PCR using the Taq man probe method in the form of absolute quantification. A series of different concentrations of leishmania were analyzed. After the purified PCR product was successfully placed in a PTG19-T plasmid vector, specialized ITS2 region was cloned in this plasmid. In the last phase, the cloned gene was transferred to the Ecoli.Top10F bacteria. The standard plasmid was provided in 10(7) to 10(1) copies/rxn concentrations. The specification and clinical sensitivity of the data was analyzed using inter and intra scales.
  
  
  RESULTS
  The probe and primer were designed using three species, including L. infantum, L. major, and L.tropica. Seven concentrations of purified parasite in culture media showed that the selected region for quantifying the parasite is suitable. Clinical and analytical specificity and sensitivity were both 100%, respectively.
  
  
  CONCLUSIONS
  The Taq man method for the ITS2 region in leishmania is one the most sensitive diagnostic test for identifying the parasite load and is suggested as a tool for fast identification and quantification of species.
authors.affiliation: Pathology and Stem Cell Research Center, Afzalipour Medical School, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Fekri Soofi Abadi
authors.affiliation: Pathology and Stem Cell Research Center, Afzalipour Medical School, Kerman University of Medical Sciences, Kerman, Iran. dabiri12@yahoo.com.
authors.forename: Shahriar
authors.initials: S
authors.lastname: Dabiri
authors.affiliation: Pathology and Stem Cell Research Center, Afzalipour Medical School, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Fotouhi Ardakani
authors.affiliation: Kerman Islamic Azad University, Kerman, Iran.
authors.forename: Lina
authors.initials: L
authors.lastname: Fani Malaki
authors.affiliation: Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Sahar
authors.initials: S
authors.lastname: Amirpoor Rostami
authors.affiliation: Pathology and Stem Cell Research Center, Afzalipour Medical School, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Mahsa
authors.initials: M
authors.lastname: Ziasistani
authors.affiliation: Pathology and Stem Cell Research Center, Afzalipour Medical School, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Donya
authors.initials: D
authors.lastname: Dabiri
mesh_terms: D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis*; D006801:Humans; D007492:Iran; D007891:Leishmania / Q000145:classification* / Q000235:genetics*; D059208:Parasite Load; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan