[BIO41] Application of PCR-RFLP technique and direct PCR-sequencing analysis to determine the relationships of Acanthamoeba

Introduction The genus of Acanthamoeba comprises of several free-living amoebae and mostly found in environment habitats worldwide. These organisms can infect a variety of mammals, including humans involving brain, eyes, skin, bones and lungs (Armstrong, 2000; Martinez, 1999). The taxonomy of Acanthamoeba is has yet to be established and still under review, although species identification of the genus by cyst morphology has been extensively used (Page, 1967). The high variability of the morphology within a clone, limited the availability of the morphology alone as a taxanomic tool (Visvesvara, 1991). New, refined and reproducible methods have been applied to resolve the taxanomy problems. The RFLP analysis was recently applied in subgenus classification of Acanthamoeba, but the result were highly polymorphic among closely related strains (Chung, et al., 1998; Kong and Chung, 1996). Comparison of high conserved sequences which have a central function like SSU rDNA gene can reveal phylogenetic relationships of organism. PCRRFLP analysis of SSU rDNA is a simpler and less expensive substitute for sequence analysis (Yu, et al., 1999). Until 1977, determining the sequence of bases in DNA sequencing was a laborious process which could only be applied to small molecules within a cloning (Innis, et al., 1990). But nowdays, direct DNA sequencing of PCR products permits the rapid characterization of sequences of interest without the need for subcloning (Newton and Graham,, 1997; Innis, et al., 1990; Biggin et al., 1983). The direct PCR Sequencing is the recent systematic studies that provided a new classification of the genus Acanthamoeba. The sequence data are very useful for identification and differentiation (Yu, et al., 1999; Stothard, et al., 1998). The present study was conducted to evaluate the PCR-DNA based techniques as a tool to distinguish relationships of Acanthamoeba spp.