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Acanthamoeba  spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of  Acanthamoeba  spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for  Acanthamoeba  spp. detection. This study improved the sensitivity of detection to avoid underestimation of  Acanthamoeba  spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of  Acanthamoeba , except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of  Acanthamoeba . The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of  Acanthamoeba  spp . , which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of  Acanthamoeba  in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic  Acanthamoeba  and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic  Acanthamoeba  spp., which is regularly found in the water resources utilised for domestic purposes.

scientific reports

Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample