Blocker-tailed PCR coupled with rolling circle amplification for fluorescent detection of emetic Bacillus cereus in milk

Abstract

Emetic Bacillus cereus (B. cereus) is a foodborne microorganism associated with vomiting disease. Herein, we have established a blocker-tailed polymerase chain reaction (bt-PCR) coupled with rolling circle amplification (RCA) assay for the sensitive and specific detection of emetic B. cereus. In this research, the designed forward primer for bt-PCR was modified into two parts, namely, the C3 spacer blocker, and the linker DNA binding sequence, which was complementary with the linker DNA. The bt-PCR products could bind to the linker DNA and initiate the RCA assay, generating numerous repetitive G-quadruplex structures. The specific fluorescence dye of thioflavin T (ThT) could bind to the G-quadruplex structures, producing enhanced fluorescent signals for detection. Under optimized conditions, the proposed assay could improve the sensitivity of the conventional PCR detection, and the limit of detection (LOD) was 2.6 × 100 CFU/mL in pure culture, and 2.8 × 100 CFU/mL in spiked milk samples with good specificity. The proposed assay showed potential application for the detection of emetic B. cereus in real world.