Engineering of a Chimeric Template Triggers RNase H-Based Isothermal Amplification Approach for Sensitive Detection of Pathogen RNA

Li Y. Zhang X. Liao Y. Shi C. Wang Y. Mu X. Xie Y. Ma C.
12 December 2023 American Chemical Society

Analytical Chemistry
2023 #95 Issue 49 18249 - 18257 pp.

RNA-based detection of pathogenic organisms is an emerging field of research that is crucial for disease diagnosis and environmental and food safety. By rationally engineering an RNA-DNA tandem (RDT) structural template, we proposed a novel RNase H-based isothermal exponential amplification (RH-IEA) reaction to rapidly identify long-stranded RNA. In this strategy, the rigid and compact RDT template selectively recognized the target RNA and formed a stable hybrid with it. Upon site-specific cleavage of RNase H, the 3′ overhang of the target RNA was cut off, and a free hydroxyl end at the hydrolysis site was generated to trigger an exponential amplification reaction (EXPAR). This method maintained the high efficiency and rapid amplification kinetics of EXPAR. As a result, the RH-IEA strategy was able to sensitively and specifically detect the characteristic sequence of Escherichia coli O157:H7 RNA, with a detection sensitivity of 1 fg/μL. Besides, the RDT template can be used as an RNA protector to prevent specific segments of the target RNA from being degraded by RNase enzymes, allowing the sample to be stored at room temperature for a long time. With this advantage, the practicality of RH-IEA will be more flexible than the reverse transcription polymerase chain reaction. It was successfully applied in the identification of E. coli O157:H7 in milk with a minimum detection concentration of 1.0 × 10² CFU/mL. Therefore, the RH-IEA method will serve as a powerful tool for detecting long-stranded RNA and will also shed light on the pathogen detection in food safety and molecular diagnosis.

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Shandong Provincial Key Laboratory of Biochemical Engineering, Qingdao Nucleic Acid Rapid Detection Engineering Research Center, Qingdao Key Laboratory of Nucleic Acid Rapid Detection, Sino-UAE International Cooperative Joint Laboratory of Pathogenic Microorganism Rapid Detection, College of Marine Science and Biological Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China
Qingdao Navid Biotechnology Co., Ltd, Qingdao JianMa Gene Technology Co., Ltd, Qingdao Nucleic Acid Rapid Testing International Science and Technology Cooperation Base, the Affiliated Hospital of Qingdao University, College of Life Sciences, Qingdao University, Qingdao, 266071, China
Clinical Laboratory, Qingdao Central Hospital, The Second Affiliated Hospital of Medical College of Qingdao University, Qingdao, 266042, China
Department of Biology, School of Sciences and Humanities, Nazarbayev University, Astana, 010000, Kazakhstan

Shandong Provincial Key Laboratory of Biochemical Engineering
Qingdao Navid Biotechnology Co.
Clinical Laboratory
Department of Biology

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