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comparison of parasite burden using real-time polymerase chain reaction assay and limiting dilution assay in leishma-nia major infected mouse
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نویسنده
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ghotloo somayeh ,mollahoseini mostafa haji ,najafi ali ,yeganeh farshid
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منبع
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iranian journal of parasitology - 2015 - دوره : 10 - شماره : 4 - صفحه:571 -576
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چکیده
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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 × 105l. 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 (pvalue = 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.
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کلیدواژه
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leishmania major ,real- time pcr ,limiting dilution assay ,balb/c mice
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آدرس
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shahid beheshti university of medical sciences, ایران, shahid beheshti university of medical sciences, ایران, pasteur institute of iran, dept of immunology, Iranf, pasteur institute of iran, dept of immunology, ایران
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پست الکترونیکی
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fyeganeh@gmail.com
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Authors
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