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Integrated silica membrane–based nucleic acid purification, amplification, and visualization platform for low-cost, rapid detection of foodborne pathogens

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Abstract

Real-time fluorescence detection of nucleic acid exhibit excellent performance in analytical and diagnostic applications. However, the requirement of laboratory-based instrument and complex nucleic acid extraction greatly limits their application in point-of-care testing (POCT). Herein, a novel integrated silica membrane–based platform incorporating nucleic acid purification, amplification, and detection steps was developed. A universal and portable visualization platform was fabricated by incorporating denaturation bubble-mediated strand exchange amplification (SEA) reaction with silica membrane. The fluorescence signal of SYBR Green I with amplification products was visualized by the naked eye using a simple ultraviolet light on the silica membrane, and significant discrimination between the positive and negative samples could be easily and visually obtained. Besides, chitooligosaccharide-modified silica membrane allows the purification of nucleic acid in a totally aqueous system and enables in situ SEA. With the proposed integrated platform, 102–108 cfu/mL Vibrio parahaemolyticus could be successfully detected and excellent performance was also revealed for gram-positive pathogens. The detection limit of the method for artificially spiked oysters was 103 cfu/g and reached 100 cfu/g after 12 h enrichment. This proof-of-concept method could also be applied to a variety of nucleic acid amplification methods. We believe that the proposed silica membrane–based platform has great potential for the rapid and low-cost detection of nucleic acids especially in low-resource settings.

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References

  1. Reta N, Saint CP, Michelmore A, Prieto-Simon B, Voelcker NH. Nanostructured electrochemical biosensors for label-free detection of water- and food-borne pathogens. ACS Appl Mater Interfaces. 2018;10(7):6055–72. https://doi.org/10.1021/acsami.7b13943.

    Article  CAS  PubMed  Google Scholar 

  2. Deng X, den Bakker HC, Hendriksen RS. Genomic epidemiology: whole-genome-sequencing–powered surveillance and outbreak investigation of foodborne bacterial pathogens. Annu Rev Food Sci Technol. 2016;7(1):353–74. https://doi.org/10.1146/annurev-food-041715-033259.

    Article  PubMed  Google Scholar 

  3. Fu Y, Zhou X, Xing D. Lab-on-capillary: a rapid, simple and quantitative genetic analysis platform integrating nucleic acid extraction, amplification and detection. Lab Chip. 2017;17(24):4334–41. https://doi.org/10.1039/C7LC01107E.

    Article  CAS  PubMed  Google Scholar 

  4. Chen J, Xu Y, Yan H, Zhu Y, Wang L, Zhang Y, et al. Sensitive and rapid detection of pathogenic bacteria from urine samples using multiplex recombinase polymerase amplification. Lab Chip. 2018;18(16):2441–52. https://doi.org/10.1039/C8LC00399H.

    Article  CAS  PubMed  Google Scholar 

  5. Strohmeier O, Marquart N, Mark D, Roth G, Zengerle R, von Stetten F. Real-time PCR based detection of a panel of food-borne pathogens on a centrifugal microfluidic “LabDisk” with on-disk quality controls and standards for quantification. Anal Methods. 2014;6(7):2038–46. https://doi.org/10.1039/C3AY41822G.

    Article  CAS  Google Scholar 

  6. Czilwik G, Messinger T, Strohmeier O, Wadle S, von Stetten F, Paust N, et al. Rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic LabDisk using highly sensitive nested PCR with integrated sample preparation. Lab Chip. 2015;15(18):3749–59. https://doi.org/10.1039/C5LC00591D.

    Article  CAS  PubMed  Google Scholar 

  7. Li J, Macdonald J. Advances in isothermal amplification: novel strategies inspired by biological processes. Biosens Bioelectron. 2015;64:196–211. https://doi.org/10.1016/j.bios.2014.08.069.

    Article  CAS  PubMed  Google Scholar 

  8. Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc. 2008;3(5):877–82. https://doi.org/10.1038/nprot.2008.57.

    Article  CAS  PubMed  Google Scholar 

  9. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):e63. https://doi.org/10.1093/nar/28.12.e63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhao Y, Chen F, Li Q, Wang L, Fan C. Isothermal amplification of nucleic acids. Chem Rev. 2015;115(22):12491–545. https://doi.org/10.1021/acs.chemrev.5b00428.

    Article  CAS  PubMed  Google Scholar 

  11. Kim T-H, Park J, Kim C-J, Cho Y-K. Fully integrated lab-on-a-disc for nucleic acid analysis of food-borne pathogens. Anal Chem. 2014;86(8):3841–8. https://doi.org/10.1021/ac403971h.

    Article  CAS  PubMed  Google Scholar 

  12. Daher RK, Stewart G, Boissinot M, Bergeron MG. Recombinase polymerase amplification for diagnostic applications. Clin Chem. 2020;62(7):947–58. https://doi.org/10.1373/clinchem.2015.245829.

    Article  CAS  Google Scholar 

  13. Compton J. Nucleic acid sequence-based amplification. Nature. 1991;350(6313):91–2. https://doi.org/10.1038/350091a0.

    Article  CAS  PubMed  Google Scholar 

  14. An L, Tang W, Ranalli TA, Kim H-J, Wytiaz J, Kong H. Characterization of a thermostable UvrD helicase and its participation in helicase-dependent amplification. J Biol Chem. 2005;280(32):28952–8. https://doi.org/10.1074/jbc.M503096200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 2004;5(8):795–800. https://doi.org/10.1038/sj.embor.7400200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Barreda-García S, Miranda-Castro R, de-los Santos-Álvarez N, Miranda-Ordieres AJ, Lobo-Castañón MJ. Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection. Anal Bioanal Chem. 2018;410(3):679–93. https://doi.org/10.1007/s00216-017-0620-3.

    Article  CAS  PubMed  Google Scholar 

  17. Shi C, Shang F, Zhou M, Zhang P, Wang Y, Ma C. Triggered isothermal PCR by denaturation bubble-mediated strand exchange amplification. Chem Commun. 2016;52(77):11551–4. https://doi.org/10.1039/C6CC05906F.

    Article  CAS  Google Scholar 

  18. Zhang M, Wang X, Han L, Niu S, Shi C, Ma C. Rapid detection of foodborne pathogen Listeria monocytogenes by strand exchange amplification. Anal Biochem. 2018;545:38–42. https://doi.org/10.1016/j.ab.2018.01.013.

    Article  CAS  PubMed  Google Scholar 

  19. Liu R, Wang X, Wang X, Shi Y, Shi C, Wang W, et al. A simple isothermal nucleic acid amplification method for the effective on-site identification for adulteration of pork source in mutton. Food Control. 2019;98:297–302. https://doi.org/10.1016/j.foodcont.2018.11.040.

    Article  CAS  Google Scholar 

  20. Rodriguez NM, Wong WS, Liu L, Dewar R, Klapperich CM. A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples. Lab Chip. 2016;16(4):753–63. https://doi.org/10.1039/C5LC01392E.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Song J, Mauk MG, Hackett BA, Cherry S, Bau HH, Liu C. Instrument-free point-of-care molecular detection of Zika virus. Anal Chem. 2016;88(14):7289–94. https://doi.org/10.1021/acs.analchem.6b01632.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rodriguez NM, Linnes JC, Fan A, Ellenson CK, Pollock NR, Klapperich CM. Paper-based RNA extraction, in situ isothermal amplification, and lateral flow detection for low-cost, rapid diagnosis of influenza a (H1N1) from clinical specimens. Anal Chem. 2015;87(15):7872–9. https://doi.org/10.1021/acs.analchem.5b01594.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Tang R, Yang H, Gong Y, You M, Liu Z, Choi JR, et al. A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection. Lab Chip. 2017;17(7):1270–9. https://doi.org/10.1039/C6LC01586G.

    Article  CAS  PubMed  Google Scholar 

  24. Byrnes SA, Bishop JD, Lafleur L, Buser JR, Lutz B, Yager P. One-step purification and concentration of DNA in porous membranes for point-of-care applications. Lab Chip. 2015;15(12):2647–59. https://doi.org/10.1039/c5lc00317b.

    Article  CAS  PubMed  Google Scholar 

  25. Hagan KA, Meier WL, Ferrance JP, Landers JP. Chitosan-coated silica as a solid phase for RNA purification in a microfluidic device. Anal Chem. 2009;81(13):5249–56. https://doi.org/10.1021/ac900820z.

    Article  CAS  PubMed  Google Scholar 

  26. Cao W, Easley CJ, Ferrance JP, Landers JP. Chitosan as a polymer for pH-induced DNA capture in a totally aqueous system. Anal Chem. 2006;78(20):7222–8. https://doi.org/10.1021/ac060391l.

    Article  CAS  PubMed  Google Scholar 

  27. Gan W, Gu Y, Han J, C-x L, Sun J, Liu P. Chitosan-modified filter paper for nucleic acid extraction and “in situ PCR” on a thermoplastic microchip. Anal Chem. 2017;89(6):3568–75. https://doi.org/10.1021/acs.analchem.6b04882.

    Article  CAS  PubMed  Google Scholar 

  28. Gan W, Zhuang B, Zhang P, Han J, Li C-X, Liu P. A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types. Lab Chip. 2014;14(19):3719–28. https://doi.org/10.1039/C4LC00686K.

    Article  CAS  PubMed  Google Scholar 

  29. Zhang Y, Zhang L, Sun J, Liu Y, Ma X, Cui S, et al. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification. Anal Chem. 2014;86(14):7057–62. https://doi.org/10.1021/ac5014332.

    Article  CAS  PubMed  Google Scholar 

  30. Zhuang B, Gan W, Wang S, Han J, Xiang G, Li C-X, et al. Fully automated sample preparation microsystem for genetic testing of hereditary hearing loss using two-color multiplex allele-specific PCR. Anal Chem. 2015;87(2):1202–9. https://doi.org/10.1021/ac5039303.

    Article  CAS  PubMed  Google Scholar 

  31. Liu C, Shi C, Li M, Wang M, Ma C, Wang Z. Rapid and simple detection of viable foodborne pathogen Staphylococcus aureus. Front Chem. 2019;7:124. https://doi.org/10.3389/fchem.2019.00124.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Guo H, Tao S. Silver nanoparticles doped silica nanocomposites coated on an optical fiber for ammonia sensing. Sensors Actuators B Chem. 2007;123(1):578–82. https://doi.org/10.1016/j.snb.2006.09.055.

    Article  CAS  Google Scholar 

  33. Tanner NA, Zhang Y, Evans TC Jr. Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. Biotechniques. 2015;58(2):59–68. https://doi.org/10.2144/000114253.

    Article  CAS  PubMed  Google Scholar 

  34. Smith C, Otto P, Bitner R, Shiels G. A silica membrane-based method for the isolation of genomic DNA from tissues and cultured cells. CSH Protoc. 2006;2006(1). https://doi.org/10.1101/pdb.prot4097.

  35. Kampeera J, Pasakon P, Karuwan C, Arunrut N, Sappat A, Sirithammajak S, et al. Point-of-care rapid detection of Vibrio parahaemolyticus in seafood using loop-mediated isothermal amplification and graphene-based screen-printed electrochemical sensor. Biosens Bioelectron. 2019;132:271–8. https://doi.org/10.1016/j.bios.2019.02.060.

    Article  CAS  PubMed  Google Scholar 

  36. Sangadkit W, Weeranoppanant N, Thipayarat A. An integrated enrichment-detection platform for identification of contamination of Vibrio parahaemolyticus in food samples. LWT Food Sci Technol. 2020;119:108841. https://doi.org/10.1016/j.lwt.2019.108841.

    Article  CAS  Google Scholar 

  37. Xing J, Yu J, Liu Y. Improvement and evaluation of loop-mediated isothermal amplification combined with chromatographic flow dipstick assays for Vibrio parahaemolyticus. J Microbiol Methods. 2020;171. https://doi.org/10.1016/j.mimet.2020.105866.

  38. Zhang M, Liu C, Shi Y, Wu J, Wu J, Chen H. Selective endpoint visualized detection of Vibrio parahaemolyticus with CRISPR/Cas12a assisted PCR using thermal cycler for on-site application. Talanta. 2020;214. https://doi.org/10.1016/j.talanta.2020.120818.

  39. Tyagi A, Saravanan V, Karunasagar I, Karunasagar I. Detection of Vibrio parahaemolyticus in tropical shellfish by SYBR green real-time PCR and evaluation of three enrichment media. Int J Food Microbiol. 2009;129(2):124–30. https://doi.org/10.1016/j.ijfoodmicro.2008.11.006.

    Article  CAS  PubMed  Google Scholar 

  40. Ahn H, Batule BS, Seok Y, Kim M-G. Single-step recombinase polymerase amplification assay based on a paper chip for simultaneous detection of multiple foodborne pathogens. Anal Chem. 2018;90(17):10211–6. https://doi.org/10.1021/acs.analchem.8b01309.

    Article  CAS  PubMed  Google Scholar 

  41. Seok Y, Joung H-A, Byun J-Y, Jeon H-S, Shin SJ, Kim S, et al. A paper-based device for performing loop-mediated isothermal amplification with real-time simultaneous detection of multiple DNA targets. Theranostics. 2017;7(8):2220. https://doi.org/10.7150/thno.18675.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Trinh TND, Lee NY. A rapid and eco-friendly isothermal amplification microdevice for multiplex detection of foodborne pathogens. Lab Chip. 2018;18(16):2369–77. https://doi.org/10.1039/C8LC00424B.

    Article  CAS  PubMed  Google Scholar 

  43. Du X-j, Zhou T-j, Li P, Wang S. A rapid Salmonella detection method involving thermophilic helicase-dependent amplification and a lateral flow assay. Mol Cell Probes. 2017;34:37–44. https://doi.org/10.1016/j.mcp.2017.05.004.

    Article  CAS  PubMed  Google Scholar 

  44. Jenison R, Jaeckel H, Klonoski J, Latorra D, Wiens J. Rapid amplification/detection of nucleic acid targets utilizing a HDA/thin film biosensor. Analyst. 2014;139(15):3763–9. https://doi.org/10.1039/C4AN00418C.

    Article  CAS  PubMed  Google Scholar 

  45. Rohrman BA, Richards-Kortum RR. A paper and plastic device for performing recombinase polymerase amplification of HIV DNA. Lab Chip. 2012;12(17):3082–8. https://doi.org/10.1039/C2LC40423K.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Choi JR, Hu J, Tang R, Gong Y, Feng S, Ren H, et al. An integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care. Lab Chip. 2016;16(3):611–21. https://doi.org/10.1039/C5LC01388G.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was financially supported by grants from the National Natural Science Foundation of China (31670868, 21675094, 31800414), Open Fund of Key Laboratory of Experimental Marine Biology, Chinese Academy of Sciences (No. KF2018NO1), Shandong Province Natural Science Foundation (ZR2019BC040), and National Key Research and Development Programs of China (2018YFE0113300).

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  1. Xiudan Wang and Chunyu Yan contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Provincial Key Laboratory of Biochemical Engineering, Qingdao Nucleic Acid Rapid Detection Engineering Research Center, College of Marine Science and Biological Engineering, Qingdao University of Science and Technology, Qingdao, 266042, Shandong, China

    Xiudan Wang, Chunyu Yan, Xiaokun Wang, Xiaoli Zhao & Cuiping Ma

  2. Qingdao Nucleic Acid Rapid Testing International Science and Technology Cooperation Base, College of Life Sciences, Department of Pathogenic Biology, School of Basic Medicine, and Department of Clinical Laboratory, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, 266071, Shandong, China

    Chao Shi

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  1. Xiudan Wang
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Wang, X., Yan, C., Wang, X. et al. Integrated silica membrane–based nucleic acid purification, amplification, and visualization platform for low-cost, rapid detection of foodborne pathogens. Anal Bioanal Chem 412, 6927–6938 (2020). https://doi.org/10.1007/s00216-020-02823-1

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