Random Amplified Polymorphic DNA Profiles as a Tool for the Identification of Acanthamoeba divionensis

Abstract

In the present study, we demonstrated the Random Amplified Polymorphism DNA (RAPD) diagnostic validity. In our study, we have analyzed RAPD profiles searching for characteristic and useful bands for Acanthamoeba diagnosis at the species level. We found a distinctive 370-bp band in A. divionensis RAPD patterns, using the OPC14 primer (TGCGTGCTTG) (Operon Technologies, Inc., Alameda, CA). The band specificity was confirmed by hybridization, using it as a probe, against all OPC14 amplifications from 10 different Acanthamoeba species. Once we sequenced this band, we used it to design a specific primer pair which showed positive amplification only in A. divionensis isolates.

Isolation of potentially pathogenic strains of Acanthamoeba in wild squirrels from the Canary Islands and Morocco

“…The DNA amplification reactions were performed, using the genus specific marker that distinguish between patho-genic and non-pathogenic Acanthamoeba strains, the extracellular serine protease related, and the A. astronyxis, A. divionensis and A. polyphaga specific primer pairs (Vodkin et al, 1992;Howe et al, 1997;Hong et al, 2000;Ortega-Rivas et al, 2003 in a 30 ll volume containing 10 ng template DNA, 50 mM Buffer KCl; 10 mM Tris-HCl, 2.5 mM MgCl 2 , 200 mM dNTP, 0.6 mM each primer, and 0.4 units of Taq DNA polymerase (Applied Biosystems, New Jersey), pH 8.3, and were performed in a Perkin-Elmer 9600 thermocycler. The cycling conditions were: an initial denaturing phase of 94 °C for 1 min and 30 repetitions at 94 °C for 1 min, annealing temperature of each primer pair for 1 min, and 72 °C for 1.5 min.…”