TY - JOUR

T1 - DNA Nanomachine (DNM) Biplex Assay for Differentiating Bacillus cereus Species

AU - Ateiah, Muhannad

AU - Gandalipov, Erik

AU - Rubel, Aleksandr A.

AU - Rubel, Maria S.

AU - Kolpashchikov, Dmitry M.

N1 - Ateiah, M.; Gandalipov, E.R.; Rubel, A.A.; Rubel, M.S.; Kolpashchikov, D.M. DNA Nanomachine (DNM) Biplex Assay for Differentiating Bacillus cereus Species. Int. J. Mol. Sci. 2023, 24, 4473. https://doi.org/10.3390/ijms24054473

PY - 2023/2/24

Y1 - 2023/2/24

N2 - 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.

AB - 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.

KW - B. cereus;

KW - ДНКзим;

KW - амплификация

KW - бинарные зонды

KW - 16S рРНК;

KW - однонуклеотидный полиморфизм

KW - B. cereus;

KW - detection of folded RNA

KW - 10–23 DNAzyme

KW - amplification-free detection

KW - binary probes

KW - 16S rRNA

KW - single nucleotide selectivity

KW - B. cereus

UR - https://www.mendeley.com/catalogue/dca5a122-a7e5-3bca-88ca-7d1096ceacde/

U2 - 10.3390/ijms24054473

DO - 10.3390/ijms24054473

M3 - Article

VL - 24

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1422-0067

IS - 5

M1 - 4473

ER -