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C. Chaumeil, P. Goldschmidt, S. Degorge, C. Saint-Jean, D. Benallaoua, L. Batellier; Detection of 10 Genotypes of Acanthamoeba Infecting Humans by Real Time PCR. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2492.
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© ARVO (1962-2015); The Authors (2016-present)
Acanthamoebae are protozoan found worldwide. Acanthamoebakeratitis (AK) has significant morbidity, often requiring corneal transplants. Central nervous system infection with Acanthamoeba is frequently fatal. The most isolated genotype from human samples is T4. Early detection and diagnosis is critical to the outcome of the clinical course of AK, however, appropriate diagnosis is often delayed and the performances of the diagnosis methods for AK are controversial due to their limited detection capacities for the different strains associated to the limited specificity (cross reactivity with bacteria, etc). The objective of this work was to design and validate a system able to detect in one step 0.1 or less cysts per µl of samples of the 10 different genotypes of Acanthamoeba by mean of a new set of primers and probe, using theFAST real-time technology that shows no cross reactivity with mammal, yeast, viral or bacterial genes.
DNA from cysts of 10 different A. genotypes were extracted with the MagNA Pure® Nucleic Acid isolation kit after 10 Min Proteinase K pre-treatment. The introduction into each sample before DNA extraction of a nonhuman calibrated virus (PhHV) served to monitor the DNA extraction and to assess PCR inhibition. PCR reactions were be carried out in 2X FAST TaqMan Mastermix® containing the forward and reverse primers (0.5 µM) + (6-FAM-AGTGTTATTCGCA TTGACTGGGTGTAA -TAMRA) probe (0.4µM) synthesised by Operon-Germany + phHV primers and probe + isolated DNA. The amplification and detection was carried out with the ABI Prism® 7500 sequence detector and cycling program consisted in one cycle at 95°C for 20 sec and 45 cycles at 95°C for 3 sec and 30 sec at 60°C.
. This technique detected the equivalent of 0.1 cyst/µl or less of Acanthamoebae in all the samples containing strains of A. hatchetti, A. tubiashi, A.astronyxis, A. lenticulata, A. comandoni, A. palestinensis ATCC-30870 and A. palestinensis ATCC-50708, A castellani, Acanthamoeba sp. ATCC-50655 and A. tubiashi.
This new broad spectrum FAST real time method is able to produce results in 2 hours and detects one or less cyst of ten different genotypes.
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