Methods and Applications: Development of a High-Throughput qPCR Assay for Detecting Waterborne Protozoa and Helminths Across Different Environmental Media in China

A One Health perspective emphasizes the close links between human, animal, and environmental health. This study selected environmental samples, including: 1) drinking water source samples from the Yangtze River in August 2023 (n=23) and Yellow River in October 2023 and January 2024 (n=23) in China (n=46); 2) sludge samples from municipal wastewater treatment plants (MWTPs) in 2015 (n=12); and 3) livestock manure in 2020 and 2021 from pigs (n=6), chickens (n=2), sheep (n=1), and cattle (n=1). These samples are closely related to human, animal, and environmental health, respectively, and were used to investigate the potential risks of pathogenic microorganisms. Studying and managing these samples will help us better understand and respond to various health risks and achieve common health goals for humans, animals, and the environment. For drinking water source samples, 10 L of water was concentrated using the calcium carbonate flocculation method, and the concentrate was transferred for DNA extraction. Sludge and livestock manure samples were used directly for DNA extraction. Environmental DNA was extracted from 0.5 g of sample concentrate using the FastDNA SPIN Kit for Soil and eluted in 75 μL of DES. Extracted DNA was stored at −20 °C until use.

Twenty-two pathogenic taxa, including 19 protozoa and 3 helminths, were selected as detection targets (Table 1). These taxa were chosen based on their waterborne characteristics and potential environmental dissemination risk as outlined in the WHO Water Quality Guidelines (4th edition) (3). Additionally, 18S rRNA and 16S rRNA genes served as quality controls. DNA was diluted to approximately 20 ng/μL, and 400 ng/μL bovine serum albumin (BSA) was added to each qPCR system to improve amplification efficiency and mitigate potential inhibition. The amplification efficiency of all reactions was between 80% and 120%.

The PCR primers and probes were designed using a Thermo Fisher proprietary process, with T_m values between 58 °C and 62 °C (12). Primer lengths were between 9 bp and 40 bp, with GC content ranging from 30% to 80%. Primer and probe information, including assay IDs, is shown in Supplementary Table S1 and stored in the Odyssey data by Thermo Fisher. The HT-qPCR assay for detecting waterborne protozoa and helminths was conducted on OpenArray chips using an Applied Biosystems QuantStudio 12K Flex instrument (Thermo Fisher, USA). The OpenArray Module enables simultaneous operation of up to four OpenArray chips, allowing testing of up to 192 samples over a 3-hour period, significantly increasing reaction throughput.

For the sensitivity test, the standard plasmid (1×10⁷ copies/μL) was diluted to a gradient of 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, 5×10², 4×10², 3×10², 2 × 10², and 1×10² copies/μL (3 replicates of each sample), and HT-qPCR assays were performed. The minimum concentration of gene copies from the standard series was considered the qPCR limit of detection (LOD). The unit of LOD was gene copies per microliter of DNA (13). For the specificity test, quality control standards of Giardia lamblia and Cryptosporidium parvum (Waterborne™, USA) were used for the qPCR assay. For the repeatability test, plasmid standards of 1×10⁵ and 1×10⁴ copies/μL were used for intra- and inter-group experiments, and the average Cycle threshold (Ct) value and coefficient of variation (CV) were calculated.

The standard plasmid (1×10⁷ copies/μL) was diluted to six gradient concentrations of 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, 5×10², and 1×10² copies/μL, with three replicates per concentration. RNase-free ddH₂O served as a negative control. The kinetic amplification curve and PCR amplification cycle threshold (Ct value) were obtained, and a standard curve was constructed by plotting the Ct value (vertical axis) against the log₁₀ (plasmid copy number) (horizontal axis). Method validity was verified based on the correlation coefficients, slopes, and amplification efficiencies of the standard curves for each detection target. Following development, the HT-qPCR assay was used to detect waterborne protozoa and helminths in various environmental samples.

Table 2 outlines the sensitivity of the HT-qPCR assay, evaluated using a gradient of positive plasmid concentrations. The results showed that all 24 detection targets were reliably detected at positive plasmid concentrations exceeding 5×10² copies/μL DNA. At concentrations of 4×10² and 3×10² copies/μL DNA, 15 and 12 targets were stably detected, respectively. When the concentration dropped below 2×10² copies/μL DNA, none of the 24 targets were consistently detected. Thus, the limit of detection for the HT-qPCR assay was established at 5×10² copies/μL DNA.

The specificity of the method was verified using standard samples of Cryptosporidium parvum and Giardia lamblia, alongside a negative control of RNase-free water. For Cryptosporidium parvum, only the qPCR assays for Cryptosporidium spp. and Cryptosporidium parvum produced positive results with Ct values less than 35 in the amplification curves, while no amplification was observed in the other assays. Similarly, for Giardia lamblia, only the qPCR assays targeting Giardia spp. and Giardia lamblia showed positive results with Ct values below 35. No positive results were observed for any of the 24 detection targets in the negative control. To assess the repeatability of the method, intra- and inter-group experiments were performed using positive plasmid concentrations of 10⁵ and 10⁴ copies/μL DNA. When the plasmid concentration was 10⁵ copies/μL DNA, the CV for intra- and inter-group experiments ranged from 1.0% to 4.1% and 1.2% to 5.5%, respectively. At 10⁴ copies/μL DNA, the CV range for intra- and inter-group experiments was 1.7%–4.6% and 3.0%–6.4%, respectively. A CV of less than 10% confirmed the method's acceptable repeatability.

A total of 24 standard curves for 19 protozoa, 3 helminths, and 18S rRNA and 16S rRNA genes were generated (Supplementary Table S1). The correlation coefficients (R²) ranged from 0.983 to 0.998, with amplification efficiencies between 80% and 107%, indicating reliable stability across all detected targets under the experimental conditions.

Table 3 presents the diagnostic performance of the HT-qPCR assay for detecting protozoa and helminths in environmental samples from drinking water sources (n=46), sludge from MWTPs (n=12), and livestock manure (n=10). In total, 17 of 22 targets were detected across all samples, with the three most prevalent being the Acanthamoeba genus (50.0%), Acanthamoeba castellanii (11.8%), and Enterocytozoon bieneusi (11.8%). The distribution of protozoa and helminths varied among the three media types. Thirteen were detected in drinking water sources, 13 in MWTP sludge, and 11 in livestock manure.

In drinking water sources, the frequently detected targets were Acanthamoeba genus, Naegleria fowleri, and Acanthamoeba castellanii, with concentrations ranging from n.d.−5.0×10⁵, n.d.−1.5×10⁵, and n.d.−4.7×10⁵ gene copies/L. In MWTP sludge, the dominant targets were Acanthamoeba genus, Isospora belli, and Acanthamoeba polyphaga, with concentrations of n.d.−7.2×10⁵, n.d.−1.2×10⁵, and n.d.−1.1×10⁵ gene copies/g, respectively. In livestock manure, Balantidium coli, Cyclospora cayetanensis, and Enterocytozoon bieneusi were detected most frequently, with concentrations of n.d.−9.9×10⁵, n.d.−4.7×10⁴, and n.d.−1.0×10⁴ gene copies/g, respectively (Supplementary Table S2). Notably, Cryptosporidium was detected more frequently than Giardia.

Importantly, Cryptosporidium spp., Enterocytozoon bieneusi, Cyclospora cayetanensis, Balantidium coli, and Isospora belli were simultaneously detected in drinking water sources, MWTP sludge, and livestock manure.

This study developed and validated an HT-qPCR assay capable of detecting 22 waterborne protozoa and helminths. This assay addresses the limitations of low throughput in existing diagnostic approaches while improving accuracy and providing a more cost-effective solution for assessing the risks of protozoa and helminths from a One Health perspective.

Compared to current detection methods, this approach significantly enhances detection efficiency. Using four OpenArray chips in a single run on the Applied Biosystems QuantStudio 12K Flex instrument, 22 targets across up to 192 samples can be analyzed within 3 hours. This rapid data generation is advantageous for large-scale monitoring and initial screening, facilitating subsequent risk assessments.

Infectious diseases caused by pathogenic bacteria, viruses, and parasites (e.g., protozoa and helminths) are the most prevalent health risks linked to drinking water. The primary threat to public health from waterborne microbes stems from drinking water contaminated with human and animal excreta (3). In this study, several parasites, including Cryptosporidium spp., Enterocytozoon bieneusi, and Cyclospora cayetanensis, were simultaneously detected in drinking water sources, MWTP sludge, and livestock manure. Animal breeding near the drinking water sources may be a potential source of contamination (14-15). For instance, waterborne transmission of Enterocytozoon bieneusi was reported during the 2013 “Pig Carcass Disposal Incident” in Shanghai, China, where dead pigs in the Huangpu River contributed to Enterocytozoon bieneusi contamination with animal and human genotypes, posing a threat to drinking water safety (16). The widespread existence of zoonotic E. bieneusi genotypes (D, EbpC, Type IV) in dogs and cats indicates that they are potential sources of environmental contamination and human infections (17). Thus, beyond Cryptosporidium and Giardia, which are already included in the National Standard for Drinking Water Quality of China (GB 5749-2022), monitoring the cross-media transmission of a broader range of protozoa and helminths is crucial for future public health management from a One Health perspective. Considering that RNA may reflect the viable pathogens in the samples, in the future, we will simultaneously quantify DNA and RNA from protozoa and helminths to improve the identification of viable pathogens (11).

This study provides a valuable tool for rapid screening and source tracking of waterborne protozoa and helminths. It is primarily used for high-throughput screening to determine the prevalence of a wide range of protozoa and helminths, characterize prevalence, and provide baseline data for identifying reference pathogens. It should be noted that the current HT-qPCR method does not allow for genotype identification of the detected pathogens. While suitable for routine monitoring, further research is needed to explore the potential for animal-environment-human transmission of waterborne pathogens.