Differentiation of Bacillus cereus Species Based on Detected Unamplified Bacterial 16S rRNA by DNA Nanomachine

Abstract

Traditional approaches for the detection and differentiation of Bacillus cereus group species often face challenges due to the complexity of genetic discrimination between species. In this protocol, we propose a simple and straightforward assay based on the detected unamplified bacterial 16S rRNA by DNA nanomachine (DNM). The assay incorporates a universal fluorescent reporter and four DNA binding fragments, three of which are responsible for "opening up" the folded rRNA while the fourth strand is responsible for detecting single nucleotide variation (SNV) with high selectivity. The binding of the DNM to 16S rRNA results in the formation of the 10-23 DNAzyme catalytic core that cleaves the fluorescent reporter and produces a signal, which is amplified over time due to catalytic turnover. The developed biplex assay enables the detection of B. thuringiensis 16S rRNA and B. mycoides at fluorescein and Cy5 channels, respectively. The protocol offers two detection options: one utilizing extracted total RNA and the other involving crude cell lysate. The latter enables a fast and straightforward detection after 1.5 h with a hands-on time of ~15 min. The new protocol 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. Key features • A sensitive and selective amplification-free biplex assay for differentiating Bacillus thuringiensis and Bacillus mycoides based on the 16S rRNA. • A simple and inexpensive assay alternative to amplification-based nucleic acid analysis, useful in environmental monitoring applications. • Adaptable for other challenging bacterial strains beyond Bacillus cereus species.

Keywords: 10-23 DNAzyme; 16S rRNA; Amplification-free detection; B. cereus; Detection of folded RNA; Single nucleotide selectivity.

Figure 1.. Design of the DNA nanomachine (DNM) used in the protocol.

(A) Design of B. thuringiensis–specific DNM. The DNM has a separate arm (Dza) and three arms (Arm 1, Arm 3, and Arm 4) attached to the dsDNA scaffold. Arms 1 and 4 were designed to tightly bind 16S rRNA, thereby unfolding its secondary structure. B. thuringiensis–specific DNM cleaves F-sub, producing fluorescent output at 525 nm. (B) The design of B. mycoides–specific DNM is similar to that of B. thuringiensis, but it cleaves Cy-Sub, producing fluorescent output at 662 nm. B. thuringiensis–specific Dza and B. mycoides–specific Dza are complementary to a fragment of 16S rRNA containing two SNVs (192 and 197). SNV 197 is used to differentiate between B. thuringiensis and B. mycoides, whereas SNV 192 is used to differentiate between the two strains from B. cereus and B. anthracis.

Figure 2.. Analysis of DNA nanomachine (DNM) associations in 12% native PAGE gel.

1: 20–100 bp ladder; 2: T1_thur strand; 3: T2_thu strand; 4: Assembled DNM.

Figure 3.. Limit of detection of synthetic DNA after 60 min of incubation at 55 °C.

Horizontal lines stand for the threshold, and vertical lines correspond to the limit of detection (LOD). The signal was read at the FAM channel. Average values of three independent measurements are presented with one standard deviation.