Development of two combined real-time quantitative OCEANII PCR assays to detect Toxoplasma gondii in amniotic fluids and sera
Abstract number: P1883
Putignani L., Russo C., Mancinelli L., Shehi E., Angelici C., Meroni V., Signore F., Adlerstein D., Menichella D.
Objectives: Toxoplasmosis is a worldwide infectious disease caused by the protozoan Toxoplasma gondii mainly acquired by feeding raw meat containing tissue cysts. Congenital infected foetuses are at risk for severe or life-threatening disease. In children, the infection may cause cervical lymphadenopathy or ocular diseases with serious clinical course. Early detection of T. gondii is therefore crucial to assess proper treatment and to avoid critical sequela. To date laboratories use customised diagnosis methods, which produce a large wide of protocols. Our research aims to provide a real-time quantitative PCR assay for rapid and sensitive detection of T. gondii in specimens.
Methods: Two real-time PCR assays, targeting internal portions of the multicopy B1 gene and repeated element RE of T. gondii, were selected to design the OCEAN II assays (DiaSorin), based on the specific hybridisation of a fluorogenic primer forming a three-way-junction with an anchor probe. In silico simulations identified optimal paired primers and thermodynamic conditions to run B1 and RE reactions. Ten-fold dilutions from 100,000 to 0.1 copy of RH strain genomic DNA was exploited, in triplicate, to titrate the dynamic range of the real-time PCR and to assess intra- and inter-assay reproducibility in more than ten independent experiments. 1 ml of sera and amniotic fluids, serologically negative for T. gondii, were spiked with 10-fold dilutions of tachyzoites from 100,000 to 0.1 in order to quantitatively correlate parasite extracted DNA and genome copies. A group of 25 amniotic fluids and sera, previously tested for T. gondii by seroconversion and PCR, were tested, as triplicates, to assay the B1- and RE-based real-time systems.
Results: The linearity of the B1 and RE assays was achieved over seven logs of input DNA, showing a consistent dynamic range and accuracy for genomic DNA in each assay (R[B1] from 0.994 to 0.999, R[RE] from 0.987 to 0.999, respectively). High inter-assay reproducibility was achieved, as inferred by R[B1]=0.998 and R[RE]=0.999. Ct values found with reference and spiked tachyzoite DNA were highly consistent for B1 and RE assays on both sera and amniotic fluids (R[B1]=0.991, R[RE]=0.990) and clinical concordance was obtained for 22 over the 25 collected samples.
Conclusions: The B1 and RE combined real-time PCR assays may represent a new diagnostic tool for rapid and quantitative detection of T. gondii in clinical specimens.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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