Abstract
There is a need for large-scale, longitudinal studies to determine the mechanisms by which the gut microbiome and its interactions with the host affect human health and disease. Current methods for profiling the microbiome typically utilize next-generation sequencing applications that are expensive, slow, and complex. Here, we present a synthetic biology platform for affordable, on-demand, and simple analysis of microbiome samples using RNA toehold switch sensors in paper-based, cell-free reactions. We demonstrate species-specific detection of mRNAs from 10 different bacteria that affect human health and four clinically relevant host biomarkers. We develop a method to quantify mRNA using our toehold sensors and validate our platform on clinical stool samples by comparison to RT-qPCR. We further highlight the potential clinical utility of the platform by showing that it can be used to rapidly and inexpensively detect toxin mRNA in the diagnosis of Clostridium difficile infections.
Highlights
There is a need for large-scale, longitudinal studies to determine the mechanisms by which the gut microbiome and its interactions with the host affect human health and disease
We demonstrate an additional potential clinical application of our RNA detection platform using the example of Clostridium difficile infection (CDI), where differentiating active infection from passive colonization has been fraught with difficulty[21]
We targeted the 16S ribosomal RNA (rRNA), because 16S rDNA profiling is a standard method for identifying bacterial species and rRNA is present at high copy numbers in bacteria
Summary
There is a need for large-scale, longitudinal studies to determine the mechanisms by which the gut microbiome and its interactions with the host affect human health and disease. We present a synthetic biology platform for affordable, on-demand, and simple analysis of microbiome samples using RNA toehold switch sensors in paper-based, cell-free reactions. We develop a method to quantify mRNA using our toehold sensors and validate our platform on clinical stool samples by comparison to RT-qPCR. Researchers often batch samples for sequencing, which can lead to significant increases in turnaround time These limitations have severely restricted the largescale prospective monitoring of patient cohorts that is necessary to provide more granular data on microbial changes and human health[15]. The second is an in vitro cell-free transcription–translation system that is freeze-dried onto paper disks for stable, long-term storage at room temperature[16]; upon rehydration, the cell-free system can execute any genetic circuit We combined these two technologies to form an abiotic platform for rapid and inexpensive development and deployment of biological sensors. Our method shows markedly different toxin mRNA expression levels in two toxigenic C. difficile strains that would otherwise be indistinguishable by standard DNAbased qPCR diagnosis
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