title: Comparison of Parasite Burden Using Real-Time Polymerase Chain Reaction Assay and Limiting Dilution Assay in Leishmania major Infected Mouse.
abstract: BACKGROUND
  Limiting dilution assay is considered as the gold standard method for quantifying the number of parasites in the animal model of Leishmania infection. Nowadays, real-time PCR is being increasingly applied to quantify infectious agents. In the present study, a real-time PCR assay was developed to estimate parasite burdens in lymph nodes of Leishmania major infected BALB/C mice. Enumeration of parasites was also performed by limiting dilution assay and compared with the results of real-time PCR based quantification.
  
  
  METHODS
  The SYBR Green based real-time PCR assay was performed to amplify a 75 bp fragment of superoxide dismutase B1 gene in the lymph nodes of L. major infected BALB/C mice 8 weeks post infection. Mice were infected subcutaneously at the base of their tail with 2 × 10(5) L. major promastigotes in the stationary phase of growth. To compare parasite burdens obtained by real-time PCR assay with those of limiting dilution assay, twelve 8-fold serial dilutions of the lymph node homogenates were prepared in the Schneider medium and incubated at 26°C. After 7 days, wells containing motile parasites were identified by direct observation under an inverted light microscope and the total number of parasites was estimated using the ELIDA software.
  
  
  RESULTS
  Spearman's correlation coefficient of the parasite burdens between real-time PCR and limiting dilution assay was 0.72 (P value = 0.008).
  
  
  CONCLUSIONS
  Real-time PCR assay is an appropriate replacement to existing limiting dilution assay in quantifying parasite burden in the experimental model of Leishmania infection.
authors.affiliation: Dept. of Immunology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Somayeh
authors.initials: S
authors.lastname: Ghotloo
authors.affiliation: Dept. of Immunology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Dept of Applied Cell Sciences, School of Advance Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Haji Mollahoseini
authors.affiliation: Dept. of Immunology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Najafi
authors.affiliation: Dept. of Immunology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Dept of Applied Cell Sciences, School of Advance Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Farshid
authors.initials: F
authors.lastname: Yeganeh
keywords: BALB/C mice; Leishmania major; Limiting dilution assay; Real- time PCR
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