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A New Method for Detecting Foodborne Pathogens Based on Argonaute Protein
2023 FFPSI Flavor Sensory Forum Scholarship
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Entry No. 001
Introduction
In May 2023, the research group of Professor Malong from Tianjin University of Science and Technology published a research paper titled “Argonaute-triggered visual and rebuilding-free foodborne pathogenic bacteria detection” in the top international journal Journal of Hazardous Materials (Q1, IF: 13.6). Li Yaru, a doctoral student at Tianjin University of Science and Technology, is the first author, and the corresponding author is Professor Malong, the Deputy Director of the Key Laboratory of Industrial Microbiology, Ministry of Education, and the National Key Laboratory of Food Nutrition and Safety.
Foodborne pathogens are the main cause of microbial contamination in food safety. In recent decades, there has been a dramatic increase in foodborne disease outbreaks due to the consumption of food contaminated with harmful bacteria, leading to high morbidity and mortality rates worldwide. Detecting harmful bacteria in food has become a crucial factor in understanding and preventing food safety and public health-related issues. Therefore, establishing a highly sensitive and rapid nucleic acid sensing platform is essential for identifying different types of foodborne pathogens. Argonaute is a programmable and target-activated nuclease that can cut invasive RNA or DNA using complementary single-stranded “guide RNA” or “guide DNA” based on Watson-Crick base pairing. Recently, due to the programmability and high specificity of Argonaute, nucleic acid detection technologies based on PfAgo (Pyrococcus furiosus Argonaute) and TtAgo (Thermus thermophilus Argonaute) have been continuously researched and established. Compared to the CRISPR/Cas system, Argonaute-mediated biosensors also have a higher specificity for nucleic acid recognition and do not rely on PAM sequence constraints. This study proposes a new method for detecting foodborne pathogens that is triggered by Argonaute and does not require reconstruction, integrating Tag-specific primer extension and exonuclease I (Exo I), called NOTE-Ago. It achieves one-step cutting detection of foodborne pathogens and has been successfully used to detect food (milk, eggs) and other complex samples for foodborne pathogen contamination.
Research Highlights
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A multifunctional biosensing platform for reconstruction-free detection of foodborne pathogens, named NOTE-Ago, has been developed.
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A portable fluorescence detection device has been designed to avoid reliance on large instruments.
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This biosensing platform can detect S. typhi and S. aureus at concentrations as low as 1 CFU/mL.
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This is the first report on developing a one-step cleavage method in Argonaute-based biosensing.
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This biosensing platform can detect other foodborne pathogens by changing the primer design.
Research Conclusions
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This study establishes a biosensing strategy based on Argonaute that integrates Tag-specific primer extension and exonuclease I (Exo I), named NOTE-Ago (Novel and One-step cleavage method based on Argonaute by integrating Tag-specific primer extension and Exonuclease I), in which Tag-specific primers amplify the invA gene of S. typhi and the nuc gene of S. aureus, and the remaining primers are digested by Exo I. The amplified products are then used as guide DNA for PfAgo. Therefore, the FAM-BHQ1 reporter probe can be cleaved by PfAgo, resulting in a change in fluorescence intensity. Through this strategy, the target nucleic acid signal can be cleverly converted into a fluorescence signal. The detection limit of NOTE-Ago biosensing can be as low as 1 CFU/mL, with a dynamic range from 1 to 108 CFU/mL. The good selectivity of NOTE-Ago biosensing further promotes its application in detecting food samples contaminated with S. typhi and S. aureus. This work enriches the detection scope based on Argonaute and provides a one-step cleavage and reconstruction-free method for ultra-sensitive detection of bacteria.
Visual Appreciation
Graphical Abstract
Figure 1. Verification and condition optimization of the feasibility of the NOTE-Ago sensing strategy. (A and B) Fluorescence spectra of each sample at an excitation wavelength of 484 nm, with an emission wavelength of 500 nm to 650 nm, showing a fluorescence peak at a wavelength of 529 nm. (C) Fluorescence images of samples taken by the fluorescence detection device and the converted grayscale photos. (D) Grayscale readings of each sample. (E) Optimization of the amount of amplified product added in the PfAgo reaction system. Optimization of PfAgo concentration (F), reporter probe concentration (G), and PfAgo reaction time in the PfAgo reaction system. (ΔFL=F−F0; F represents the fluorescence intensity measured after the reaction, F0 represents the background fluorescence intensity. “+” indicates the presence of target or Exo I, “-” indicates the absence of target or Exo I).
Figure 2. Schematic diagram of the structure of the 3D-fluorescence detection device
Figure 3. Schematic diagram of the internal structure of the 3D-fluorescence detection device
Figure 4. Performance analysis of NOTE-Ago biosensing. (A and B) Fluorescence spectral analysis of samples of different concentrations of S. typhi and S. aureus at an excitation wavelength of 484 nm and an emission wavelength of 500 nm to 650 nm. (C) Fluorescence readings of different concentrations of S. typhi and S. aureus samples at an emission wavelength of 529 nm. (D) Selectivity analysis of the proposed NOTE-Ago method for detecting S. typhi and S. aureus. Concentrations of S. typhi, S. aureus, and interference cells were 108 CFU/mL. (ΔFL=F−F0; F represents the fluorescence intensity measured after the reaction, F0 represents the background fluorescence intensity).
Figure 5. Comparison of the detection capabilities of NOTE-Ago biosensing and qPCR for detecting S. typhi and S. aureus contaminated samples. (A and B) Analysis of S. typhi detected by NOTE-Ago method, SBYR Green method, and TaqMan method. (C) ROC curve analysis of detection results for S. typhi. (D and E) Analysis of S. aureus detected by NOTE-Ago method, SBYR Green method, and TaqMan method. (F) ROC curve analysis of detection results for S. aureus. (ΔFL=F−F0; F represents the fluorescence intensity measured after the reaction, F0 represents the background fluorescence intensity).
Figure 6. Detection of S. typhi and S. aureus in orange juice using NOTE-Ago biosensing. (A) Fluorescence readings of detection results of S. typhi and S. aureus in orange juice samples. (B) Fluorescence images of S. typhi (a) and S. aureus (b) in orange juice samples taken by the fluorescence detection device. (C) Grayscale readings of detection results of S. typhi and S. aureus in orange juice samples. (D) Converted grayscale photos of S. typhi (a) and S. aureus (b). (E and F) Detection results of S. typhi and S. aureus in orange juice samples using plate counting method. (ΔFL=F−F0; F represents the fluorescence intensity measured after the reaction, F0 represents the background fluorescence intensity).
Figure 7. Detection of S. typhi and S. aureus in eggs using NOTE-Ago biosensing. (A) Fluorescence readings of detection results of S. typhi and S. aureus in egg samples. (B) Fluorescence images of S. typhi (a) and S. aureus (b) in egg samples taken by the fluorescence detection device. (C) Grayscale readings of detection results of S. typhi and S. aureus in egg samples. (D) Converted grayscale photos of S. typhi (a) and S. aureus (b). (E and F) Detection results of S. typhi and S. aureus in egg samples using plate counting method. (ΔFL=F−F0; F represents the fluorescence intensity measured after the reaction, F0 represents the background fluorescence intensity).
Malong
Malong (Corresponding Author), male, born in 1983, obtained a PhD from the University of Edinburgh in the UK, and has conducted postdoctoral research at the University of St Andrews in the UK. He is currently a professor at the School of Biological Engineering, Tianjin University of Science and Technology, a doctoral supervisor, and the Deputy Director of the Key Laboratory of Industrial Microbiology, Ministry of Education, and the National Key Laboratory of Food Nutrition and Safety. In the past five years, he has successively hosted the National Natural Science Foundation Youth and General Projects (2 projects); the Tianjin Overseas High-level Talent Introduction Project; the Tianjin Science and Technology Plan Project; the Tianjin Natural Science Foundation; and the Tianjin Key Research and Development Project. In recent years, he has published nearly 80 SCI papers, of which 72 were published in I and II zone journals. The total impact factor of the articles is >560, with 55 papers having an impact factor >5 and 16 papers having an impact factor >10; the total citation of the articles is nearly 2000 times, with an H-index >25. He mainly engages in research on the detection of food hazards and the bioactivity of foodborne natural products. He has received several honors such as the “Overseas High-level Talent Introduction” in Tianjin in 2015, the first tier of the “131” Innovation Talent in Tianjin in 2018, the “Young and Middle-aged Science and Technology Innovation Leading Talent” in the Tianjin Innovation Talent Promotion Plan in 2019, the National Silver Award in the first National Postdoctoral Innovation and Entrepreneurship Competition in 2021, the “Tianjin Youth May Fourth Medal” in 2022, and the “Outstanding Scientific and Technological Worker in Tianjin” in 2022.
Li Yaru
Li Yaru, a doctoral student in the 2020 class of Light Industry Technology and Engineering at the School of Biological Engineering, Tianjin University of Science and Technology. Her research direction is food safety detection, mainly focusing on the construction and application of nucleic acid and nuclease-based biosensors, under the supervision of Professor Malong. She has published 8 SCI articles as the first author in top international journals such as Trends in Biotechnology (IF 21.942), Comprehensive Reviews in Food Science and Food Safety (IF 15.786), and Journal of Hazardous Materials (IF 14.224), of which 5 were published in JCR I and II zone journals, with a total impact factor greater than 70, including 3 papers with an impact factor greater than 10, and has applied for 3 patents. She has hosted and completed one Tianjin University graduate research innovation project and participated in one National Natural Science Foundation general project, two Tianjin Natural Science Foundation general projects, and contributed to two books. She won the National Scholarship for Doctoral Students in 2022, the first-class academic scholarship twice and the second-class academic scholarship once at Tianjin University of Science and Technology, and has received the title of “Outstanding Graduate Cadre” three times, “Top Ten Academic Stars” at Tianjin University of Science and Technology, and the honorary title of “Jinnan Anti-epidemic Warrior,” and participated as a speaker in the 12th “Bohai Wind” Graduate Academic Culture Season “Thinking and Acting” Master-Doctor Academic Innovation Forum.
Original Link
https://doi.org/10.1016/j.jhazmat.2023.131485
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