DNA Nanomachine (DNM) Biplex Assay for Differentiating Bacillus cereus Species

Abstract

Conventional methods for the detection and differentiation of Bacillus cereus group species have drawbacks mostly due to the complexity of genetic discrimination between the Bacillus cereus species. Here, we describe a simple and straightforward assay based on the detected unamplified bacterial 16S rRNA by DNA nanomachine (DNM). The assay uses a universal fluorescent reporter and four all-DNA binding fragments, three of which are responsible for “opening up” the folded rRNA while the fourth stand is responsible for detecting single nucleotide variation (SNV) with high selectivity. Binding of the DNM to 16S rRNA results in the formation of the 10–23 deoxyribozyme catalytic core that cleaves the fluorescent reporter and produces a signal, which is amplified over time due to catalytic turnover. This developed biplex assay enables the detection of B. thuringiensis 16S rRNA at fluorescein and B. mycoides at Cy5 channels with a limit of detection of 30 × 103 and 35 × 103 CFU/mL, respectively, after 1.5 h with a hands-on time of ~10 min. The new assay may simplify the analysis of biological RNA samples and might be useful for environmental monitoring as a simple and inexpensive alternative to amplification-based nucleic acid analysis. The DNM proposed here may become an advantageous tool for detecting SNV in clinically significant DNA or RNA samples and can easily differentiate SNV under broadly variable experimental conditions and without prior amplification.

DNAzyme Nanomachine with Fluorogenic Substrate Delivery Function: Advancing Sensitivity in Nucleic Acid Detection

“…The results showed that HDNM is highly selective as it did not produce significant fluorescence even when incubated with high concentrations of the nonspecific Influenza A virus for 3 h at 55 °C (Figure ). This high selectivity is consistent with prior studies on binary probes based on deoxyribozyme 10–23, − among other binary probes, ,− proving their excellent selectivity, which is relevant to their applications in single-nucleotide polymorphisms (SNP) genotyping, and it makes it a promising candidate for virus detection and diagnosis, contributing significantly to public health efforts.…”