An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for Listeria monocytogenes detection

作者：Li, Fan; Ye, Qinghua; Chen, Moutong; Zhou, Baoqing; Zhang, Jumei; Pang, Rui; Xue, Liang; Wang, Juan; Zeng, Haiyan; Wu, Shi; Zhang, Youxiong; Ding, Yu^*; Wu, Qingping^*

来源：Biosensors and Bioelectronics, 2021, 179: 113073.

DOI：10.1016/j.bios.2021.113073

• CRISPR biosensor
• Electrochemical biosensor
• Listeria monocytogenes
• Cas12a
• Nucleic acid detection
• Point-of-care

Listeria monocytogenes is an important foodborne pathogen that can cause listeriosis with high patient mortality. Accordingly, it is necessary to develop a L. monocytogenes detection platform with high specificity, sensitivity, and exploitability. CRISPR/Cas systems have shown great potential in the development of next-generation biosensors for nucleic acid detection, owing to the trans-cleavage capabilities of the Cas effector proteins. Herein, we introduce the trans-cleavage activity of CRISPR/Cas12a into an electrochemical biosensor (ECRISPR), combined with recombinase-assisted amplification (RAA), to establish a cost-effective, specific and ultrasensitive method; namely RAA-based E-CRISPR. The concept behind this approach is that the target will induce the number change of the surface signaling probe (containing an electrochemical tag), which leads to a variation in the electron transfer of the electrochemical tag. The introduction of an RAA-based Cas12a system into the E-CRISPR sensor achieves a more prominent signal change between the presence and absence of the target. Under optimized conditions, RAA-based E-CRISPR can detect as low as 0.68 aM of genomic DNA and 26 cfu/mL of L. monocytogenes in pure cultures. More importantly, the RAA-based E-CRISPR enables rapid and ultrasensitive detection of L. monocytogenes in spiked and natural Flammulina velutipes samples. Moreover, no cross-reactivity with other non-target bacteria was observed. This system thus demonstrates to be a simple, high sensitivity, and high-accuracy platform for L. monocytogenes detection.

• 单位
  广东省微生物研究所; 华南农业大学; y