Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Rapid Detection of Streptococcus mutans Using an Integrated Microfluidic System with Loop-Mediated Isothermal Amplification

  • Jingfu Wang (State Key Laboratory of Military Stomatology and National Clinical Research Center for Oral Diseases & Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture, Department of Craniofacial Trauma and Orthognathic Surgery, School of Stomatology, The Fourth Military Medical University) ;
  • Jingyi Wang (College of Information and Electrical Engineering, Shenyang Agricultural University) ;
  • Xin Chang (Outpatient Department, The Ninth Retired Cadres Retreat of Liaoning Military Command) ;
  • Jin Shang (Department of Stomatology, General Hospital of Northern Theater Command) ;
  • Yuehui Wang (Department of Stomatology, General Hospital of Northern Theater Command) ;
  • Qin Ma (State Key Laboratory of Military Stomatology and National Clinical Research Center for Oral Diseases & Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture, Department of Craniofacial Trauma and Orthognathic Surgery, School of Stomatology, The Fourth Military Medical University) ;
  • Liangliang Shen (Department of Biochemistry and Molecular Biology, Fourth Military Medical University)
  • Received : 2023.04.17
  • Accepted : 2023.05.16
  • Published : 2023.08.28
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Abstract

Streptococcus mutans is the primary causative agent of caries, which is one of the most common human diseases. Thus, rapid and early detection of cariogenic bacteria is critical for its prevention. This study investigated the combination of loop-mediated isothermal amplification (LAMP) and microfluid technology to quantitatively detect S. mutans. A low-cost, rapid microfluidic chip using LAMP technology was developed to amplify and detect bacteria at 2.2-2.2 × 106 colony-forming units (CFU)/ml and its detection limits were compared to those of standard polymerase chain reaction. A visualization system was established to quantitatively determine the experimental results, and a functional relationship between the bacterial concentration and quantitative results was established. The detection limit of S. mutans using this microfluidic chip was 2.2 CFU/ml, which was lower than that of the standard approach. After quantification, the experimental results showed a good linear relationship with the concentration of S. mutans, thereby confirming the effectiveness and accuracy of the custom-made integrated LAMP microfluidic system for the detection of S. mutans. The microfluidic system described herein may represent a promising simple detection method for the specific and rapid testing of individuals at risk of caries.

Keywords

Acknowledgement

This research was funded by the Department of Science and Technology of Liaoning Province, China (grant number: 2022-BS-175). The funding agency had no role in the collection, analysis and interpretation of data, writing of the report, or in the decision to submit the article for publication.

References

  1. Li Y, Saraithong P, Chen Z, Leung E, Pattanaporn K, Dasanayake A. 2017. Comparison of real-time quantitative PCR with a chairside test for Streptococcus mutans assessment. Chin. J. Dent. Res. 20: 199-210. 
  2. Loesche WJ. 1986. Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50: 353-380.  https://doi.org/10.1128/mr.50.4.353-380.1986
  3. Bai G, Tian Y, Wu J, Gu Y, Chen Z, Zeng F, Liu J. 2019. Construction of a fusion anti-caries DNA vaccine in transgenic tomato plants for PAcA gene and cholera toxin B subunit. Biotechnol. Appl. Biochem. 66: 924-929.  https://doi.org/10.1002/bab.1806
  4. Loesche WJ, Straffon LH. 1979. Longitudinal investigation of the role of Streptococcus mutans in human fissure decay. Infect. Immun. 26: 498-507.  https://doi.org/10.1128/iai.26.2.498-507.1979
  5. Beighton D, Russell RR, Whiley RA. 1991. A simple biochemical scheme for the differentiation of Streptococcus mutans and Streptococcus sobrinus. Caries Res. 25: 174-178.  https://doi.org/10.1159/000261363
  6. Hamada S, Slade HD. 1980. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol. Rev. 44: 331-384.  https://doi.org/10.1128/mr.44.2.331-384.1980
  7. Smorawinska M, Kuramitsu HK. 1992. DNA probes for detection of cariogenic Streptococcus mutans. Oral Microbiol. Immunol. 7: 177-181.  https://doi.org/10.1111/j.1399-302X.1992.tb00532.x
  8. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579: 270-273.  https://doi.org/10.1038/s41586-020-2012-7
  9. Lazcka O, Del Campo FJ, Munoz FX. 2007. Pathogen detection: a perspective of traditional methods and biosensors. Biosens. Bioelectron. 22: 1205-1217.  https://doi.org/10.1016/j.bios.2006.06.036
  10. Kim J, Jung C. 2022. SF-qPCR: strand displacement-based fast quantitative polymerase chain reaction. BioChip J. 16: 41-48.  https://doi.org/10.1007/s13206-021-00044-x
  11. Vincent M, Xu Y, Kong H. 2004. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5: 795-800.  https://doi.org/10.1038/sj.embor.7400200
  12. Yao C, Zhang R, Tang J, Yang D. 2021. Rolling circle amplification (RCA)-based DNA hydrogel. Nat. Protoc. 16: 5460-5483. [  https://doi.org/10.1038/s41596-021-00621-2
  13. Choi G, Jung JH, Park BH, Oh SJ, Seo JH, Choi JS, et al. 2016. A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria. Lab Chip 16: 2309-2316.  https://doi.org/10.1039/C6LC00329J
  14. Jang M, Kim S. 2022. Inhibition of non-specific amplification in loop-mediated isothermal amplification via tetramethylammonium chloride. BioChip J. 16: 326-333.  https://doi.org/10.1007/s13206-022-00070-3
  15. Cao G, Li J, Xing Z, Zhang Z, Zhang W, Li C, et al. 2021. Establishment of scalable nanoliter digital LAMP technology for the quantitative detection of multiple myeloproliferative neoplasm molecular markers. Sens. Actuators B 346: 130493.
  16. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28: E63. 
  17. Kim S, Kim JH, Kim S, Park JS, Cha BS, Lee ES, et al. 2022. Loop-mediated isothermal amplification-based nucleic acid lateral flow assay for the specific and multiplex detection of genetic markers. Anal. Chim. Acta 1205: 339781. 
  18. Hu YQ, Huang XH, Guo LQ, Shen ZC, Lv LX, Li FX, et al. 2021. Rapid and visual detection of Vibrio parahaemolyticus in aquatic foods using blaCARB-17 gene-based loop-mediated isothermal amplification with lateral flow dipstick (LAMP-LFD). J. Microbiol. Biotechnol. 31: 1672-1683.  https://doi.org/10.4014/jmb.2107.07022
  19. Nagamine K, Kuzuhara Y, Notomi T. 2002. Isolation of single-stranded DNA from loop-mediated isothermal amplification products. Biochem. Biophys. Res. Commun. 290: 1195-1198.  https://doi.org/10.1006/bbrc.2001.6334
  20. Nagamine K, Hase T, Notomi T. 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell. Probes 16: 223-229.  https://doi.org/10.1006/mcpr.2002.0415
  21. Park S, Zhang Y, Lin S, Wang TH, Yang S. 2011. Advances in microfluidic PCR for point-of-care infectious disease diagnostics. Biotechnol. Adv. 29: 830-839.  https://doi.org/10.1016/j.biotechadv.2011.06.017
  22. Drese KS. 2019. Lab on a chip, Internist (Berl.). German 60: 339-344.  https://doi.org/10.1007/s00108-018-0526-y
  23. Huang G, Huang Q, Xie L, Xiang G, Wang L, Xu H, Ma L, Luo X, Xin J, Zhou X, Jin X. 2017. A rapid, low-cost, and microfluidic chip-based system for parallel identification of multiple pathogens related to clinical pneumonia. Sci. Rep. 7: 6441. 
  24. Terry SC, Jerman JH, Angell JB. 1979. A gas chromatographic air analyzer fabricated on a silicon wafer. IEEE Trans. Electron. Devices 26: 1880-1886.  https://doi.org/10.1109/T-ED.1979.19791
  25. Dittrich PS, Tachikawa K, Manz A. 2006. Micro total analysis systems. Latest advancements and trends. Anal. Chem. 78: 3887-3908.  https://doi.org/10.1021/ac0605602
  26. Fang X, Liu Y, Kong J, Jiang X. 2010. Loop-mediated isothermal amplification integrated on microfluidic chips for point-of-care quantitative detection of pathogens. Anal. Chem. 82: 3002-3006.  https://doi.org/10.1021/ac1000652
  27. Van Nguyen H, Nguyen VD, Lee EY, Seo TS. 2019. Point-of-care genetic analysis for multiplex pathogenic bacteria on a fully integrated centrifugal microdevice with a large-volume sample. Biosens. Bioelectron. 136: 132-139.  https://doi.org/10.1016/j.bios.2019.04.035
  28. Herr AE, Hatch AV, Giannobile WV, Throckmorton DJ, Tran HM, Brennan JS, et al. 2007. Integrated microfluidic platform for oral diagnostics. Ann. N. Y. Acad. Sci. 1098: 362-374.  https://doi.org/10.1196/annals.1384.004
  29. Li Z, Ju R, Sekine S, Zhang D, Zhuang S, Yamaguchi Y. 2019. All-in-one microfluidic device for on-site diagnosis of pathogens based on an integrated continuous flow PCR and electrophoresis biochip. Lab. Chip. 19: 2663-2668.  https://doi.org/10.1039/C9LC00305C
  30. Hunt RWG, Pointer MR. 2011. Measuring Colour, 4th Ed. John Wiley & Sons, Ltd., Hoboken, New Jersey. 
  31. Joiner A. 2004. Tooth colour: a review of the literature. J. Dent. 32: 3-12.  https://doi.org/10.1016/j.jdent.2003.10.013
  32. O'Brien WJ, Hemmendinger H, Boenke KM, Linger JB, Groh CL. 1997. Color distribution of three regions of extracted human teeth. Dent. Mater. 13: 179-185.  https://doi.org/10.1016/S0109-5641(97)80121-2
  33. Fischbach J, Xander NC, Frohme M, Glokler JF. 2015. Shining a light on LAMP assays--a comparison of LAMP visualization methods including the novel use of berberine. Biotechniques 58: 189-94.  https://doi.org/10.2144/000114275
  34. He Y, Xie T, Tong Y. 2021. Rapid and highly sensitive one-tube colorimetric RT-LAMP assay for visual detection of SARS-CoV-2 RNA. Biosens. Bioelectron. 187: 113330. 
  35. Bau HH, Liu C, Mauk M, Song J. 2017. Is instrument-free molecular detection possible? Expert Rev. Mol. Diagn. 17: 949-951.  https://doi.org/10.1080/14737159.2017.1374855
  36. Nam D, Kim S, Kim JH, Lee S, Kim D, Son J, et al. 2023. Low-temperature loop-mediated isothermal amplification operating at physiological temperature. Biosensors (Basel). 13(3),367. [doi: 10.3390/bios13030367]. 
  37. Xia L, Zhang H, Lu Y, Cai J, Wang B, Jian J. Development of a loop-mediated isothermal amplification assay for rapid detection of Nocardia salmonicida, the causative agent of nocardiosis in fish. J. Microbiol. Biotechnol. 25: 321-327.