Multiplex bacterial detection in CRISPR/Cas13a droplet fluids
High-throughput detection of low-level bacteria is critical in public health, food safety, and first response. In this paper, we present for the first time a droplet microfluidics-based platform with recombinase-assisted amplification (RAA)-assisted one-pot clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 13a (CRISPR /Cas13a) analysis and droplet encoding strategies for accurate and sensitive nucleic acid determination of various foodborne pathogens.
This workflow takes full advantage of CRISPR/Cas13a signal amplification and droplet confinement effects to improve detection sensitivity and enable endpoint quantification. At the same time, by changing the color of the droplets, the number of bacteria detected simultaneously is greatly increased. It is capable of detecting seven different types of foodborne pathogens simultaneously. It is worth noting that this system was also applied to actual food samples with satisfactory results. Overall, given the advantages in high sensitivity, outstanding selectivity, and large-scale multiplexing, the one-pot CRISPR/Cas13a-based droplet microfluidic system can be expanded and generalized for the identification of other bacteria.
Review of droplet microfluidic systems based on one-pot CRISPR/Cas13a
(A) Concept of one-pot CRISPR/Cas13a analysis. Bacteria can be detected using a single-step reaction in which RAA generates dsDNA from target DNA, which is then translated into RNA to activate cleavage of a Cas13a-based fluorescent reporter gene in a single step.
(B) Schematic diagram of coded droplet generation. Solutions of different target primers were emulsified with oil together with food dyes into thousands of coded droplets and further mixed to form a droplet library.
(C) Workflow of CRISPR/Cas13a-based droplet microfluidic system. Target-containing droplets are generated and electrode actuation is used to elicit responses that randomly merge with encoded droplets. After incubation, single-layer droplet arrays were imaged brightly and fluorescently to identify and quantify target bacteria.
Construction and optimization of one-pot CRISPR/Cas13a assays
(A) Fluorescence analysis of feasibility of one-pot CRISPR/Cas13a assay.
(B) Agarose gel electrophoresis analysis of Cas13a-mediated cleavage.
(C) Heat map analysis of fluorescence results of specific assays. optimization
(D) FQ5U reporting concentration,
(E) Temperature and (F) incubation time.
Schematic diagram of droplet encoding and decoding
(A) Construction process of droplet encoding strategy.
(B) RGB color space differences of droplets with 95% confidence interval.
(C) Image and size distribution of droplets generated by the droplet generation chip.
(D) Color-coded droplets are collected and decoded using a Matlab script.
(E) Percentage of false positives and false negatives for color recognition.
Characterization of microelectrode modules
(A) Process of fabricating microelectrodes by injecting liquid solder into microchannels.
(B) Microscope images of droplets under triggered and non-triggered merging conditions.
(C) Analysis of droplet size distribution before and after merging.
(D) Adjust electrode voltage to initiate droplet merging. Error bars represent standard deviation of pooled diameters
Evaluation of droplet microfluidic systems for bacterial detection
(A) Linear relationship between measured concentration (Y) and exceptional concentration (X). Inserts are representative fluorescence images of microarrays using DNA concentration serial dilutions of ST for single-pot CRISPR/Cas13a assays.
(B) Specificity test of droplet microfluidic system targeting bacteria. (n = 3, error bars represent changes in measured DNA copy number).
(C) Heat map showing determined DNA concentrations in spike lettuce by digital one-pot CRISPR/Cas13a and qPCR analysis.
(D) Performance results of digital one-pot CRISPR/Cas13a in detecting 8 food samples, showing 100% consistency with qPCR results.
