Abstract
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.
Highlights
Quantitative molecular diagnostic methods, such as quantitative polymerase chain reaction, can target nucleic acid gene sequences specific to known microbial pathogens
cycle quantification (Cq) values across replicate chips were highly repeatable for synthetic DNA standards (R2 = 0.989, Figure 1A), for synthetic DNA in a complex stool DNA matrix (R2 = 0.984, Figure 1B) and for DNA extracted from fecal samples collected from children in Bangladesh (R2 = 0.935, Figure 1C)
Fecal samples displayed a median difference in Cq values of 0.39 [IQR 0.15– 0.81] (Figure 1C) across all assays, which corresponds to a coefficient of variation on calculated gene copy number of 28% [IQR 16–50] (Table 2, Repeatability)
Summary
Quantitative molecular diagnostic methods, such as quantitative polymerase chain reaction (qPCR), can target nucleic acid gene sequences specific to known microbial pathogens. These methods have provided insights in the study of diarrheal disease beyond what can be gained using microbiological cell culture or immunoassays (van den Berg et al, 2007; Liu et al, 2016b; PlattsMills et al, 2018) and have been applied successfully in the field of pathogen detection for decades (Wood et al, 1994; Lin et al, 2000; He et al, 2002). The per-sample cost of the enteric TAC is $60, not including labor, capital equipment, or DNA extraction reagents (Liu et al, 2013)
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