comparison of parasite burden using real-time polymerase chain reaction assay and limiting dilution assay in leishma-nia major infected mouse

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). conclusion: real-time pcr assay is an appropriate replacement to existing limit­ing dilution assay in quantifying parasite burden in the experimental model of leishma­nia infection.