The Culture And Identification Of 18S Ribosomal DNA Genotype Of Acanthamoeba From Patients With Keratitis

PURPOSE:To establish the economic and effective methods of isolation and culture of the Acanthamoeba sp which isolated from patients with keratitis;To identify the genotype of 18S ribosomal RNA gene(18S rDNA, Rns) of the Acanthamoeba strains;And to discuss the feasibility and superiority of early diagnosis of acanthamoeba Keratitis using related technologies.METHODS:The corneal scrapings from the ulcers of patients para- diagnosed AK were transferred to non-nutrient agar (NNA) plates covered with a 24-hour-old culture of E. coli. The plates were sealed and incubated at 28°C for 14days, then got the single cyst planted on a new NNA and examined for amoeba growth with the inverted microscope. We used a hot-initiated PCR-based method with the acanthamoeba- specific primers JDP1 and JDP2 to detect acanthamoeba. Partial genome DNA sequences of 18SrDNA of Acanthamoeba were amplified, gel electrophoresis observe . another epithetlial specimen which was observed with 10% KOH under microscope as the reference .Washed the superficies of NNA and then extracted the genome DNA of acanthamoeba,the same way to amplify partial genome DNA. The PCR products were sequenced. The partial DNA sequences were analyzed by software Clustal X and MEGA2.RESULTS:1,Culture of acanthamoeba : On the non-nutrient agar (NNA) plates covered with a 24-hour-old culture of E. coli., 28℃raises about 3 days, has the acanthamoeba growth. Take the inoculation sites assumes radiated as the center. 14 cases (78%) were culture-positive keratitis.2,Result of PCR: The corneal scrapings from the ulcers of patients para- diagnosed AK to remove direct PCR amplification. 16 cases (18 eyes), one of 12 cases (67%) can observe the Acanthamoeba-specific band. the fragment about 450bp. This way can Confirmed Acanthamoeba parasite infection.3,Corneal Scraping test under microscope: 5 eyes found acanthamoeba follicle by observing the epithelial specimen with 10 % KOH under microscope.With the Fisher exact propability to add up the two diagnostic method,and P<0.05. United PCR technology and corneal Scraping test under microscope can improve diagnosis rate of Acanthamoeba keratitis to 83.3%.4,The Rns genotype classification results:Of all 14 DF3 sequences obtained from The 14 corneal specimens of culture-positive , a total of 8 gene sequence different from each other, but all of them were Rns genotype T4.CONCLUSIONS:The experiment methods of isolation and culture of the Acanthamoeba have been established. All Acanthamoeba strains which isolated from keratitis were Rns genotype T4, and 50 percent come from water pollution. Polymerase chain reaction of 18SrDNA has important clinical value in diagnose of AK early and its classification clearly.