Highly Sensitive Determination of Bacillus subtilis by Microchip Electrophoresis Combining With A Dual Nucleic Acid Recycling Amplification

In dairy products, Bacillus subtilis (B. subtilis) is considered a harmful spoilage bacterium. Consequently, it is imperative to establish highly sensitive and selective approaches for detecting B. subtilis. In this article, quantification of B. subtilis via its 16S rRNA was first performed by microchip electrophoresis (MCE) combined with a dual nucleic acid recycling amplification strategy involving apyrimidinic endonuclease 1 (APE1)-mediated cycle and catalytic hairpin assembly (CHA). In APE1-mediated cycle, two specially designed probes containing apurinic/apyrimidinic sites (AP sites) capture B. subtilis 16S rRNA, and then APE1 cleaves the AP sites to release the recycled target RNA. Next, the product of APE1-mediated cycle triggers the CHA and generates the final product for further MCE detection. Under the optimal conditions, the limit of detection for B. subtilis was 29 CFU/mL (S/N = 3). The proposed strategy was successfully applied to the detection of B. subtilis in pasteurized milk and exhibited high speed, high sensitivity, and good specificity.