Professor Li Boan's team developed a one-step nucleic acid rapid detection technology based on CRISPR/Cas12a that is not restricted by PAM

In recent decades, respiratory infectious diseases caused by pathogens Bordetella pertussis (BP), influenza virus, SARS-CoV-2, etc. have seriously threatened human health, especially the outbreak of coronavirus disease 2019 (COVID-19) in 2019. The rapid, early and accurate detection of nucleic acids is of great significance for the diagnosis of diseases, the evaluation of treatment options, the formulation of preventive measures, the evaluation of drug resistance, etc., which in turn can prevent the risk of disease transmission.

‍‍‍‍‍Since its inception, CRISPR has been widely used in molecular diagnosis due to its rapidity, sensitivity, strong specificity, and constant reaction temperature, such as the SHERLOCK nucleic acid detection platform established by Zhang Feng's team, and the DETECTR detection technology established by Jennifer's team. However, for these CRISPR/Cas-based detection strategies, the design of crRNA is limited by the protospacer adjacent motif site (PAM) sequence, and usually needs to be carried out in steps, requiring the nucleic acid to be pre-amplified first, and then the amplified The product is transferred to the CRISPR/Cas system for nucleic acid detection, a multi-step reaction method that is not only cumbersome and time-consuming, but also susceptible to aerosol contamination. In order to avoid the problem of uncapped contamination, Li Boan's team combined CRISPR technology with microfluidic chips in the early stage to automate the reaction and avoid additional pipetting steps ((Sun et al., Science China-Chemistry, 2022). Recently, research In further research, the team developed a one-step nucleic acid detection technology that is not limited by PAM. Constant temperature amplification and gene editing detection are carried out in the same reaction tube without product transfer steps, which is simple, convenient and highly sensitive. The results were published online in ANALYTICA CHIMICA ACTA as a research paper entitled "Fast and visual detection of nuclear acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM".‍

‍‍‍In this study, by studying the cleavage activity of CRISPR/Cas12a to recognize PAM and non-PAM targets, it was found that compared with the recognition of PAM targets, the ability of Cas12a to recognize cis-cleaved target templates of non-PAM targets was significantly weakened, but trans-cleaved single-stranded DNA probes ability remains unchanged. The research team then combined CRISPR/Cas12a with RPA constant temperature amplification technology to detect 64 random PAM (TTTN) targets in one step. , TNT) showed poor performance in ORCD, while TCC, TCG, CCT, CTC, GTC, and CTG showed excellent activity in ORCD. Further research found that reducing the cis cleavage activity of CRISPR/Cas12a increased the sensitivity of CRISPR to detect PAM targets by 1000 times, which means that cis cleavage activity is the key to CRISPR one-step detection.‍‍

Using non-PAM sequences, the CRISPR one-step method has fast detection speed, high sensitivity and strong specificity, and single-copy DNA and RNA detection can be achieved within 30 minutes. And combined with the nucleic acid extraction-free technology, the ORCD system can complete the extraction, amplification and detection of samples within 30 minutes. In clinical applications, 82 clinical samples of Bordetella pertussis were detected. Compared with PCR, the sensitivity and specificity They are 97.30% and 100% respectively. In addition, RT-ORCD detected 13 SARS-CoV-2 samples, and the results were consistent with RT–PCR. And the results can be seen with only a portable UV lamp or blue light. This nucleic acid detection method, which is not limited by PAM, one-step, fast, result visualization, high sensitivity and specificity, shows great potential in the future development of POCT molecular diagnosis in the post-pandemic era. It can be widely used to quickly detect infectious diseases in non-professional settings, such as small clinics, airports, fields and other places.

‍The first author of the paper is Lin Kangfeng, a doctoral student from the School of Life Sciences of Xiamen University, Guo Jianguang, a doctoral student, and Guo Xiangju, a master’s student, are the co-first authors of this paper. Professor Li Boan from the School of Life Sciences of Xiamen University, Dr. Zhang Rui from the First Affiliated Hospital of Professor Wu Jinzhun from the Municipal Maternal and Child Health Hospital is the co-corresponding author of this article. This project was supported by the National Key Research and Development Program (2020YFA0112300), the National Natural Science Foundation of China (81972458, 32071150), and the Fujian Provincial Health Education Joint Research Project (2019-WJ-34).

Paper link: https://doi.org/10.1016/j.aca.2023.340938(Photo/Text Li Boan Team)