Quantification of Acanthamoeba spp. and Hartmannella vermiformis by real-time polymerase chain reaction
Date Issued
2008
Date
2008
Author(s)
Wu, Ying-Chieh
Abstract
Free-living amoebae (FLA) of Acanthamoeba spp. and Hartmannella vermiformis are widely distributed in various aquatic habitats. They are the hosts of many pathogenic bacteria and have been found to cause opportunistic infection. In this study, we developed a real-time quantitative polymerase chain reaction (real-time qPCR) to target these two types of amoebae. For selecting a better DNA extraction kit, we compared two commercial DNA extraction kits, Wizard® SV genomic DNA purification system (Promega) and FastDNA spin kit for soil (MP).We optimized each protocol of these two kits at first, and the data showed it was better when using microcentrifuge method for Promega and increasing both vortex time and elution volume for MP. Both kits were further coupled with real-time qPCR assay to determine the DNA quantity of Acanthamoeba spp. and H. vermiformis from the samples prepared in lab and collected from water, substrate biofilm (SB) or floating biofilm (FB) of cooling towers. After 100-fold, 100-fold, and 10-fold dilution of DNA extracted from water, SB, and FB samples, respectively, and determination by real-time qPCR, quantity of Acanthamoeba DNA extracted by MP was 0.7, 0.5, and 0.8 log fg, respectively, more than that by Promega. Similarity, H. vermiformis DNA extracted by MP was also 1.3, 1.1, and 1.4 log fg greater than that by Promega, respectively. Moreover, the number of amoebae-positive samples was generally greater in MP-extracted samples than that in Promega-extracted ones regardless of sample type or amoebic genera, indicating MP is more applicable for environmental samples than Promega. We further adopted MP to extract DNA from trophozoites and cysts of A. castellanii and H. vermiformis to evaluate the effect of pretreatment in water and FB samples on DNA quantity. With the DNA extracted from the sample without pretreatment as reference, the relative DNA recovery rates of water and FB samples were 88.10±8.48% and 65.93±10.96% for A. castellanii trophozoite, respectively, and 110.09±22.95% and 94.69±7.25% for A. castellanii cyst, respectively. As for H. vermiformis, the respective recovery rates of water and FB samples were 108.14±13.08% and 75.07±31.24% for trophozoite and 137.15±72.25% and 78.31±17.04% for cyst. These results indicate the DNA recovery rate of water samples was greater than that of FB samples for both of A. castellanii and H. vermiformis. Finally, a calibration curve between amoebic number and DNA quantity was determined, from which the detection limit was found as low as 3 amoebae. In conclusion, the present study showed MP kit coupled with appropriate DNA dilution and real-time qPCR assay is an optimal method to quantify Acanthamoeba spp. and H. vermiformis from artificial water reservoirs. This methodology provides the potential to accurately characterize the distribution of these two types of amoebae, and may be further used to evaluate the relationship between the amoebae and their parasitic pathogens.
Subjects
DNA extraction
real-time quantitative polymerase chain reaction
Type
thesis
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