Wash-Free and High-Contrast Imaging of Single-Nucleotide Variation in Single Cells Using Transcription-Amplified Light-Up RNA Aptamer.

Single-nucleotide variation (SNV) imaging can indicate cellular heterogeneity and spatial pattern, but it remains challenging to produce high-gain signal while also yielding single-nucleotide resolution. Herein, we developed a light-up strategy for visualizing SNVs based on transcription amplification, enabling wash-free and high-contrast imaging of SNVs inside cells. The discrimination of SNVs is achieved by ligase-assisted transcription reaction. Employing a light-up RNA aptamer as a reporter eliminates nonspecific probe binding and the washing process and contributes to a 2-fold improvement of signal gain compared to that using the fluorescence in situ hybridization (FISH) method. The method allowed us to precisely quantify drug-resistant strains in the bacteria mixture and identify drug-resistant Salmonella enterica (S. enterica) isolated from poultry farm. Using this approach, we explored the colonization features of drug-resistant and drug-sensitive S. enterica in the mice intestinal tract and screened the prebiotics for Salmonella colonization inhibition. The SNV imaging method promises for the interrogation of genotypes in physiological and pathological states at the single-cell level.

Rapid and Sensitive Detection of Fungicide-Resistant Crop Fungal Pathogens Using an Isothermal Amplification Refractory Mutation System.

Fungicide abuse leads to the emergence of fungicide-resistant fungal pathogens, thus posing a threat to agriculture and food safety. Here, we developed an isothermal amplification refractory mutation system (termed iARMS) allowing us to resolve genetic mutations, enabling rapid, sensitive, and potentially field-applicable detection of fungicide-resistant crop fungal pathogens. iARMS yielded a limit of detection of 25 aM via a cascade signal amplification strategy of recombinase polymerase amplification (RPA) and Cas12a-mediated collateral cleavage at 37 °C within 40 min. Specificity for fungicide-resistant Puccinia striiformis (P. striiformis) detection was guaranteed by RPA primers and the flexible sequence of gRNA. The iARMS assay allowed us to detect as low as 0.1% cyp51-mutated P. striiformis that showed resistance to the demethylase inhibitor (DMI), which was 50 times more sensitive than the sequencing techniques. Thus, it is promising for the discovery of rare fungicide-resistant isolates. We applied iARMS to investigate the emergence of fungicide-resistant P. striiformis in western China and found that its proportion was over 50% in Qinghai, Sichuan, and Xinjiang Province. iARMS can serve as a molecular diagnostic tool for crop diseases and facilitate precision plant disease management.