农村生活污水处理厂中阿米巴原虫的定量检测

Quantitative detection of amebic protozoa from rural domestic wastewater treatment plant

【目的】自由生活的棘阿米巴属(Acanthamoeba spp.)和哈曼属原虫(Hartmannella vermiformis)普遍存在于自然界的土壤和各种水体中,这两个属中的某些种类被认为对人和动物具有潜在的致病性,应用染料法实时荧光定量PCR技术建立特异性强、灵敏度高及重复性好的快速检测阿米巴虫的方法具有实际意义。【方法】采用非培养方法选择适合低拷贝基因检测的荧光染料BRYT Green?dye用于农村生活污水处理厂不同工艺阶段水样中Acanthamoeba spp.和H.Vermiformis 18S r RNA基因的检测和定量分析。【结果】在整个处理工艺流程中均检测到Acanthamoeba spp.和H.Vermiformis,并呈现出不同的变化趋势,进水中分别达到8.70×105拷贝/L和1.84×106拷贝/L。与进水相比,调节池、好氧池和膜池中阿米巴原虫的数量均降低了1-2个数量级,但是出水中Acanthamoeba spp.则出现增加趋势。【结论】对阿米巴原虫可能造成的潜在健康危害应引起重视,并有必要作为污水处理达标的补充标准。

[Objective] Acanthamoeba spp. and Hartmannella vermiformis are free-living amoebae （FLA）, which are widely ubiquitous in soils and various aquatic environments. Certain species in these two genus were recognized as potential opportunistic pathogenicity for human and animals. It has practical significance of developing a highly specific, sensitive and reproducible assay which based on real-time fluorescence quantitative PCR for identifying amoebae. [Methods] We employed a culture-independent method, the real-time quantitative PCR assays to detect 18S rRNA genes ofAeanthamoeba spp. and H. vermiformis using BRYT Green dye in different processes of rural domestic wastewater treatment plant. [Results] The results indicated that the most frequently amoebae discovered were Acanthamoeba spp. and H. vermiformis which appear in all treatment system stages. The concentrations of two amoebae genus were up to 8.70× 10^5 and 1.84× 10^6 copies/L in untreated wastewater, respectively. Compared with untreated wastewater, the abundance of amoebae in adjusting tank, aerobic tank and membrane tank decreased in one or two orders of magnitude, while Acanthamoeba spp. was one order of magnitude higher in treated wastewater than untreated wastewater. [Conclusion] Hence, it should be paid much attention to amoebae having health risk and it is necessary to use amoebae as an additional reference standard for domestic wastewater treatment.