Waterborne pathogens are threatening public health globally, but profiling multiple human pathogenic bacteria (HPBs) in various polluted environments is still a challenge due to the absence of rapid, high-throughput and accurate quantification tools. This work developed a novel chip, termed the HPB-Chip, based on high-throughput quantitative polymerase chain reactions (HT-qPCR). The HPB-Chip with 33-nL reaction volume could simultaneously complete 10,752 amplification reactions, quantifying 27 HPBs in up to 192 samples with two technical replicates (including those for generating standard curves). Specific positive bands of target genes across different species and single peak melting curves demonstrated high specificity of the HPB-Chip. The mixed plasmid serial dilution test validated its high sensitivity with the limit of quantification (LoD) of averaged 82 copies per reaction for 25 target genes. PCR amplification efficiencies and R coefficients of standard curves of the HPB-Chip averaged 101 % and 0.996, respectively. Moreover, a strong positive correlation (Pearson’ : 0.961–0.994, < 0.001) of HPB concentrations (log copies/L) between HPB-Chip and conventional qPCR demonstrated high accuracy of the HPB-Chip. Subsequently, the HPB-Chip has been successfully applied to absolutely quantify 27 HPBs in municipal and hospital wastewater treatment plants (WWTPs) after PMA treatment. A total of 17 HPBs were detected in the 6 full-scale WWTPs, with an additional 19 in the hospital WWTP. Remarkably, , and were present in the final effluent of each municipal WWTP. Overall, the HPB-Chip is an efficient and accurate high-throughput quantification tool to comprehensively and rapidly quantify 27 HPBs in the environment.

中文翻译：

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HPB-Chip：一种基于高通量 qPCR 的准确工具，用于快速分析环境中水生人类致病菌


水传播病原体正在威胁全球公共卫生，但由于缺乏快速、高通量和准确的定量工具，对各种污染环境中的多种人类致病菌 (HPB) 进行分析仍然是一个挑战。这项工作开发了一种基于高通量定量聚合酶链式反应 (HT-qPCR) 的新型芯片，称为 HPB-Chip。反应体积为 33 nL 的 HPB-Chip 可同时完成 10,752 个扩增反应，通过两次技术重复（包括用于生成标准曲线的重复）对多达 192 个样品中的 27 个 HPB 进行定量。不同物种的目标基因的特定阳性条带和单峰熔解曲线证明了 HPB 芯片的高度特异性。混合质粒连续稀释测试验证了其高灵敏度，25 个靶基因的定量限 (LoD) 为每次反应平均 82 个拷贝。 HPB-Chip的PCR扩增效率和标准曲线R系数平均分别为101%和0.996。此外，HPB-Chip 和传统 qPCR 之间的 HPB 浓度（log 拷贝数/L）呈强正相关（Pearson'：0.961–0.994，< 0.001），这证明了 HPB-Chip 的高精度。随后，HPB-Chip 已成功应用于对 PMA 处理后的市政和医院废水处理厂 (WWTP) 中的 27 种 HPB 进行绝对定量。 6 个正式污水处理厂共检测到 17 种 HPB，其中医院污水处理厂检测到 19 种 HPB。值得注意的是， 、 和 都存在于每个城市污水处理厂的最终废水中。总体而言，HPB-Chip是一种高效、准确的高通量定量工具，可全面、快速地量化环境中的27种HPB。