Recently, Professor Feng Yan’s team from the National Key Laboratory of Microbial Metabolism at Shanghai Jiao Tong University published a research paper titled “Thermal activation of Argonaute nuclease enables one-pot multiplex detection of viruses” in the internationally renowned journal Sensors and Actuators B: Chemical. This research developed a one-pot nucleic acid detection system compatible with amplification and enzyme digestion based on thermally activated immobilized Argonaute enzyme. PhD student Ye Xingyu from the School of Life Science and Technology at Shanghai Jiao Tong University is the first author, with Professor Feng Yan and Associate Researcher Liu Qian as co-corresponding authors. Shanghai Jiao Tong University is the primary completion unit, with Jianghong Biotechnology (Shanghai) Co., Ltd. and Shanghai Children’s Medical Center as co-completion units.
Figure 1 Reagent Development Route
Figure 2 Reagent and Equipment Model
Based on CRISPR/Cas and Argonaute (Ago) programmable nucleases, nucleic acid detection technology has advantages of being rapid, portable, and sensitive, overcoming the limitations of isothermal amplification that cannot perform multiplex detection and the non-specific amplification. However, as programmable nucleases interfere with amplification, two-step reactions of amplification and enzyme digestion are usually required. Nevertheless, two-step reactions can easily lead to aerosol contamination of amplification products and are relatively cumbersome to operate. Therefore, developing a one-pot detection system compatible with amplification and enzyme digestion is crucial for the clinical application of programmable nucleases.
The Ago from thermophilic archaea has higher thermal stability than Cas proteins, making it suitable for developing thermally activated immobilized enzymes, which means that Ago activity is blocked during the low-temperature amplification phase and released during the high-temperature enzyme digestion phase. First, the study confirmed that the ability of Argonaute to bind ssDNA and dsDNA is the main reason for amplification obstruction, and verified that the immobilized Ago microspheres have no significant effect on the efficiency of LAMP isothermal amplification.
Subsequently, the research team optimized the compatibility of the amplification-enzyme digestion system regarding metal ions, pyrophosphatase, immobilized enzyme particle size, and enzyme quantity, establishing the OPTIMAL (One-Pot Test on IMmobilized Argonaute and LAMP) one-pot detection system. This system achieved sensitivity consistent with commercial qPCR kits (5 copies/μL) in the detection of the novel coronavirus and enabled at-home visualization detection on handheld devices.
Figure 3 Program and Product Features
In August 2023, WHO tuberculosis prevention and control expert Professor Madhukar Pai mentioned in the Chinese Journal of Antituberculosis: In the global movement to eliminate tuberculosis, “decentralized instant testing must complement existing centralized laboratory testing.” The article specifically noted that China’s low-cost products and prevention and control programs are suitable for global promotion for tuberculosis prevention and elimination. The article also mentioned that “pathogen detection technology should shift from single disease detection to multi-disease detection (with the versatility and affordability of molecular detection devices allowing for the implementation of multiple infectious disease tests);” this is key to enhancing the current capabilities of grassroots and frontline clinical diagnosis and treatment. This research was supported by the National Natural Science Foundation and the Shanghai Municipal Science and Technology Commission – Shanghai Jiao Tong University’s Basic Research Special Plan. Thanks to Professor Song Jinzhhao from the Hangzhou Institute of Medicine/ Zhejiang Cancer Hospital for assistance in home testing, and to the National Translational Medicine Research Center for data analysis support.
Figure 4 Application of OPTIMAL in Home Testing
Paper link:
https://www.sciencedirect.com/science/article/pii/S0925400524003162?dgcid=coauthor
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