Quantitative molecular diagnostic methods can effectively detect pathogen-specific nucleic acid sequences, but costs associated with multi-pathogen panels hinder their widespread use in research trials. Nano-liter qPCR (nL-qPCR) is a miniaturized tool for quantification of multiple targets in large numbers of samples based on assay parallelization on a single chip, with potentially significant cost-savings due to rapid throughput and reduced reagent volumes. We evaluated a suite of novel and published assays to detect 17 enteric pathogens using a commercially available nL-qPCR technology. Amplification efficiencies ranged from 88 to 98% (mean 91%) and were reproducible across four operators at two separate facilities. When applied to fecal material, assays were sensitive and selective (99.8% of DNA amplified were genes from the target organism). Due to nanofluidic volumes, detection limits were 1–2 orders of magnitude less sensitive for nL-qPCR than an enteric TaqMan Array Card (TAC). However, higher detection limits do not hinder detection of diarrhea-causing pathogen concentrations. Compared to TAC, nL-qPCR displayed 99% (95% CI 0.98, 0.99) negative percent agreement and 62% (95% CI 0.59, 0.65) overall positive percent agreement for presence of pathogens across diarrheal and non-diarrheal fecal samples. Positive percent agreement was 89% among samples with concentrations above the nL-qPCR detection limits. nL-qPCR assays showed an underestimation bias of 0.34 log₁₀ copies/gram of stool [IQR −0.40, −0.28] compared with TAC. With 12 times higher throughput for a sixth of the per-sample cost of the enteric TAC, the nL-qPCR chip is a viable alternative for enteropathogen quantification for studies where other technologies are cost-prohibitive.

定量分子诊断方法可以有效地检测病原体特异性核酸序列，但与多病原体检测相关的成本阻碍了它们在研究试验中的广泛应用。纳升qPCR（nL-qPCR）是一种微型工具，可基于单个芯片上的化验平行化对大量样品中的多个目标进行定量，由于快速通量和减少的试剂量，可显着节省成本。我们评估了一套新颖的检测方法，并使用市售的nL-qPCR技术检测了17种肠道病原体。放大效率从88％到98％（平均91％）不等，并且可以在两个单独的工厂的四个操作员之间再现。当应用于粪便材料时，测定法既灵敏又选择性（扩增出的DNA的99.8％是来自目标生物的基因）。由于纳米流体的体积，nL-qPCR的检测限度比肠溶性TaqMan阵列卡（TAC）低1-2个数量级。但是，较高的检测限不会妨碍检测引起腹泻的病原体浓度。与TAC相比，nL-qPCR在整个腹泻和非腹泻粪便样本中均显示出99％（95％CI 0.98，0.99）阴性百分率和62％（95％CI 0.59，0.65）总体阳性百分率。在浓度高于nL-qPCR检测限的样品中，阳性一致性百分比为89％。nL-qPCR分析显示低估偏差为0.34 log 较高的检测限不会妨碍检测引起腹泻的病原体浓度。与TAC相比，nL-qPCR在整个腹泻和非腹泻粪便样本中均显示出99％（95％CI 0.98，0.99）阴性百分率和62％（95％CI 0.59，0.65）总体阳性百分率。在浓度高于nL-qPCR检测限的样品中，阳性一致性百分比为89％。nL-qPCR分析显示低估偏差为0.34 log 较高的检测限不会阻碍检测引起腹泻的病原体浓度。与TAC相比，nL-qPCR在整个腹泻和非腹泻粪便样本中均显示出99％（95％CI 0.98，0.99）阴性百分率和62％（95％CI 0.59，0.65）总体阳性百分率。在浓度高于nL-qPCR检测限的样品中，阳性一致性百分比为89％。nL-qPCR分析显示低估偏差为0.34 log与TAC相比，粪便[IQR -0.40，-0.28]为₁₀拷贝/克。nL-qPCR芯片的通量提高了12倍，而肠道TAC的单样本成本仅为后者的六分之一，因此它是肠病原体定量研究的可行替代品，可用于其他成本不高的研究。