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A real-time dPCR system was developed to improve the sensitivity, specificity and quantification accuracy of end point dPCR. We compared three technologies - real-time qPCR, end point dPCR and real-time dPCR - in the context of SARS-CoV-2. Some improvement in limit of detection was obtained with end point dPCR compared with real-time qPCR, and the limit of detection was further improved with the newly developed real-time dPCR technology through removal of false-positive signals. Real-time dPCR showed increased linear dynamic range compared with end point dPCR based on quantitation from amplification curves. Real-time dPCR can improve the performance of TaqMan assays beyond real-time qPCR and end point dPCR with better sensitivity and specificity, absolute quantification and a wider linear range of detection.

The human microbiome encompasses a variety of microorganisms that change dynamically and are in close contact with the body. The microbiome influences health and homeostasis, as well as the immune system, and any significant change in this equilibrium (dysbiosis) triggers both acute and chronic health conditions. Microbiome research has surged, in part, due to advanced sequencing technologies enabling rapid, accurate, and cost-effective identification of the microbiome. A major prerequisite for stool sample collection to study the gut microbiome in longitudinal prospective studies requires standardized protocols that can be easily replicated. However, there are still significant bottlenecks to stool specimen collection that contribute to low patient retention rates in microbiome studies. These barriers are further exacerbated in solid organ transplant recipients where diarrhea is estimated to occur in up to half the patient population. We sought to test two relatively easy sample collection methods (fecal swab and wipes) and compare them to the more cumbersome "gold" standard collection method (scoop) using two different sequencing technologies (16S ribosomal RNA sequencing and shotgun metagenomics). Our comparison of the collection methods shows that both the swabs and the wipes are comparable to the scoop method in terms of bacterial abundance and diversity. The swabs, however, were closer in representation to the scoop and were easier to collect and process compared to the wipes. Potential contamination of the swab and the wipe samples by abundant skin commensals was low in our analysis. Comparison of the two sequencing technologies showed that they were complementary, and that 16S sequencing provided enough coverage to detect and differentiate between bacterial species identified in the collected samples. Our pilot study demonstrates that alternative collection methods for stool sampling are a viable option in clinical applications, such as organ transplant studies. The use of these methods may result in better patient retention recruitment rates in serial microbiome studies.