Introduction:The establishment of a highthroughput quantification approach for waterborne pathogenic protozoa and helminths is crucial for rapid screening and health risk assessment.Methods:We developed a high-through...Introduction:The establishment of a highthroughput quantification approach for waterborne pathogenic protozoa and helminths is crucial for rapid screening and health risk assessment.Methods:We developed a high-throughput quantitative polymerase chain reaction(HT-qPCR)assay targeting 19 waterborne protozoa and 3 waterborne helminths and validated its sensitivity,specificity,and repeatability.The assay was then applied to test various environmental media samples.Results:The HT-qPCR assay's limit of detection(LOD)was 5×10^(2) copies/μL DNA,and its specificity was confirmed using Giardia and Cryptosporidium standards.Repeatability,assessed through intra-and inter-group experiments,yielded a coefficient of variation(CV)of 1.0%–4.6%and 1.2%–6.4%at concentrations of 1×10^(5) and 1×10^(4) copies/μL,respectively.The R^(2) values of the 22 standard curves ranged from 0.983 to 0.998,with amplification efficiencies between 80%and 107%.In drinking water sources,sludge from municipal wastewater treatment plants(MWTPs),and livestock manure samples,17 of 22 targets were detected,with Acanthamoeba genus(50.0%),Acanthamoeba castellanii(11.8%),and Enterocytozoon bieneusi(11.8%)showing high prevalence.Cryptosporidium spp.,Enterocytozoon bieneusi,and Cyclospora cayetanensis were simultaneously found in all three sample types.Discussion:This study presents a useful tool for the rapid detection of waterborne protozoa and helminths in complex environmental microbiomes,providing scientific data for monitoring cross-media transmission and controlling microbial risk from a One Health perspective.展开更多
基金Supported by the National Key Research and Development Program of China(2022YFC3204703)the China Postdoctoral Innovation Talents Support Program(BX20230400).
文摘Introduction:The establishment of a highthroughput quantification approach for waterborne pathogenic protozoa and helminths is crucial for rapid screening and health risk assessment.Methods:We developed a high-throughput quantitative polymerase chain reaction(HT-qPCR)assay targeting 19 waterborne protozoa and 3 waterborne helminths and validated its sensitivity,specificity,and repeatability.The assay was then applied to test various environmental media samples.Results:The HT-qPCR assay's limit of detection(LOD)was 5×10^(2) copies/μL DNA,and its specificity was confirmed using Giardia and Cryptosporidium standards.Repeatability,assessed through intra-and inter-group experiments,yielded a coefficient of variation(CV)of 1.0%–4.6%and 1.2%–6.4%at concentrations of 1×10^(5) and 1×10^(4) copies/μL,respectively.The R^(2) values of the 22 standard curves ranged from 0.983 to 0.998,with amplification efficiencies between 80%and 107%.In drinking water sources,sludge from municipal wastewater treatment plants(MWTPs),and livestock manure samples,17 of 22 targets were detected,with Acanthamoeba genus(50.0%),Acanthamoeba castellanii(11.8%),and Enterocytozoon bieneusi(11.8%)showing high prevalence.Cryptosporidium spp.,Enterocytozoon bieneusi,and Cyclospora cayetanensis were simultaneously found in all three sample types.Discussion:This study presents a useful tool for the rapid detection of waterborne protozoa and helminths in complex environmental microbiomes,providing scientific data for monitoring cross-media transmission and controlling microbial risk from a One Health perspective.
