For rDNA typing of isolates as well as for diagnostic assay, the PCRswere done in 20-µL reactions using 1 and 3 µL of template DNA(for amoebae and corneal scraping samples, respectively). Each PCR contained1 U of Taq DNA polymerase (AmpliTaq Gold; PerkinElmer,Inc, Boston, Mass), 2pM of each primer, 200µM dNTPs (deoxyribose nucleotidetriphosphates), and 1.5mM magnesium chloride and was amplified for 35 three-stepcycles of 94°C, 61°C, and 72°C, each for 1 minute, followed byfinal extension of 72°C for 5 minutes in an MJR PTC-200 thermocycler (MJResearch Inc, Waltham, Mass). In each PCR amplification, the cycling profilewas preceded by one heating step of 94°C for 10 minutes to activate the Taq DNA polymerase. To develop a direct gel-based assayfor Acanthamoeba detection in clinical samples, primersfor the 2 rDNA targets were tested individually and also in combination, initiallywith DNA from culture-proved and "Type" isolates of Acanthamoeba and then with the use of DNA from 34 corneal scraping samples frompatients with keratitis. The amplified products were resolved and visualizedon ethidium bromide–stained 1.5% agarose gel. In all the experiments,proper negative (water in place of template DNA) and positive (DNA from Acanthamoeba castellanii, ATCC 50370) controls were used.Moreover, specificity of the assay was tested with DNA from 2 Acanthamoeba-related protozoa, Balamuthia mandrillaris and Hartmanella vermiformis (ATCC 30966),and human leukocytes, Pseudomonas aeruginosa, Aspergillus species, and herpes simplex virus. The sensitivitywas determined by testing 10-fold dilutions of A castellanii DNA.