Advertisement

Analytical and Bioanalytical Chemistry

, Volume 411, Issue 3, pp 647–658 | Cite as

Improvement and evaluation of loop-mediated isothermal amplification combined with a chromatographic flow dipstick assay and utilization in detection of Vibrio cholerae

  • Jia Yu
  • Feixue Wang
  • Xijing Zhan
  • Xin Wang
  • Feng Zuo
  • Yuxi Wei
  • Jun QiEmail author
  • Yin LiuEmail author
Research Paper

Abstract

Loop-mediated isothermal amplification (LAMP) is a specific, sensitive, and easy-to-perform nucleic acid analytical technique with wide application for diagnosis of disease. Recently, LAMP combined with use of a lateral chromatographic flow dipstick (LFD) has been widely used in nucleic acid detection. However, the LFD mechanism has not been systematically analyzed, and the optimal combination of labeled primers has not been adequately evaluated. We analyzed the LAMP mechanism and discovered that the labeled loop primers played a significant role in the LFD assay. To verify our hypothesis, we developed two LFD assays for Vibrio cholerae to detect the ctxA gene and the 16S–23S ribosomal DNA internal transcribed spacer (ITS). We labeled the inner primers [forward inner primer (FIP) and backward inner primer (BIP)] and loop primers [forward loop primer (LF) and backward loop primer (LB)]. Then the labeled and unlabeled primers were combined to form ten different primer sets. We assessed the specificity, sensitivity, and efficiency of LFD assays with use of different primer compositions. All triple-labeled primer sets resulted in false positive results in the LFD assay, as did the FIP and BIP double-labeled primer set. Other double-labeled-primer sets used in LFD assays showed higher sensitivity than the LAMP assays. Moreover, FIP and LF double-labeled and BIP and LB double-labeled sets had the highest sensitivity. In both cases, assays could be performed in 20 min. We also applied the ITS LFD assays in food samples. The enrichment broths of 112 oyster samples were tested, and the proportion that tested positive by the LFD assays was 6.25%, which was not lower than the rate for the conventional PCR method (5.36%).

Graphical abstract

Keywords

Loop-mediated isothermal amplification Chromatographic flow dipstick ctx16S–23S ribosomal DNA internal transcribed spacer Vibrio cholerae 

Notes

Acknowledgements

This work was supported by a project from the National Undergraduate Training Program for Innovation and Entrepreneurship (no. 201510055113).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

References

  1. 1.
    Huq A, Haley BJ, Taviani E, Chen A, Hasan NA, Colwell RR. Detection, isolation, and identification of Vibrio cholerae from the environment. Curr Protoc Microbiol. 2012;26:6A.5.1–51.  https://doi.org/10.1002/9780471729259.mc06a05s26.Google Scholar
  2. 2.
    Ramamurthy T, Garg S, Sharma R, Bhattacharya SK, Nair GB, Shimada T, et al. Emergence of novel strain of Vibrio cholerae with epidemic potential in southern and eastern India. Lancet. 1993;341(8846):703–4.CrossRefGoogle Scholar
  3. 3.
    Yamazaki W, Seto K, Taguchi M, Ishibashi M, Inoue K. Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification. BMC Microbiol. 2008;8:94.  https://doi.org/10.1186/1471-2180-8-94.CrossRefGoogle Scholar
  4. 4.
    Bliem R, Schauer S, Plicka H, Obwaller A, Sommer R, Steinrigl A, et al. A novel triplex quantitative PCR strategy for quantification of toxigenic and nontoxigenic Vibrio cholerae in aquatic environments. Appl Environ Microbiol. 2015;81(9):3077–85.  https://doi.org/10.1128/AEM.03516-14.CrossRefGoogle Scholar
  5. 5.
    Yamazaki W. Sensitive and rapid detection of Campylobacter jejuni and Campylobacter coli using loop-mediated isothermal amplification. Methods Mol Biol. 2013;943:267–77.  https://doi.org/10.1007/978-1-60327-353-4_18.CrossRefGoogle Scholar
  6. 6.
    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.CrossRefGoogle Scholar
  7. 7.
    Chen Y, Cheng N, Xu Y, Huang K, Luo Y, Xu W. Point-of-care and visual detection of P. aeruginosa and its toxin genes by multiple LAMP and lateral flow nucleic acid biosensor. Biosens Bioelectron. 2016;81:317–23.  https://doi.org/10.1016/j.bios.2016.03.006.CrossRefGoogle Scholar
  8. 8.
    Ahmed MU, Saito M, Hossain MM, Rao SR, Furui S, Hino A, et al. Electrochemical genosensor for the rapid detection of GMO using loop-mediated isothermal amplification. Analyst. 2009;134(5):966–72.  https://doi.org/10.1039/b812569d.CrossRefGoogle Scholar
  9. 9.
    Borysiak MD, Kimura KW, Posner JD. NAIL: nucleic acid detection using isotachophoresis and Lloop-mediated isothermal amplification. Lab Chip. 2015;15(7):1697–707.  https://doi.org/10.1039/c4lc01479k.CrossRefGoogle Scholar
  10. 10.
    Nagatani N, Yamanaka K, Saito M, Koketsu R, Sasaki T, Ikuta K, et al. Semi-real time electrochemical monitoring for influenza virus RNA by reverse transcription loop-mediated isothermal amplification using a USB powered portable potentiostat. Analyst. 2011;136(24):5143–50.  https://doi.org/10.1039/c1an15638a.CrossRefGoogle Scholar
  11. 11.
    Xu W, Wang C, Zhu P, Guo T, Xu Y, Huang K, et al. Real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons. Analyst. 2016;141(8):2542–52.  https://doi.org/10.1039/c6an00145a.CrossRefGoogle Scholar
  12. 12.
    Jaroenram W, Kiatpathomchai W, Flegel TW. Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick. Mol Cell Probes. 2009;23(2):65–70.  https://doi.org/10.1016/j.mcp.2008.12.003.CrossRefGoogle Scholar
  13. 13.
    Choi JR, Hu J, Gong Y, Feng S, Wan Abas WA, Pingguan-Murphy B, et al. An integrated lateral flow assay for effective DNA amplification and detection at the point of care. Analyst. 2016;141(10):2930–9.  https://doi.org/10.1039/c5an02532j.CrossRefGoogle Scholar
  14. 14.
    Waters RA, Fowler VL, Armson B, Nelson N, Gloster J, Paton DJ, et al. Preliminary validation of direct detection of foot-and-mouth disease virus within clinical samples using reverse transcription loop-mediated isothermal amplification coupled with a simple lateral flow device for detection. PLoS One. 2014;9(8):e105630.  https://doi.org/10.1371/journal.pone.0105630.CrossRefGoogle Scholar
  15. 15.
    Rigano LA, Marano MR, Castagnaro AP, Do Amaral AM, Vojnov AA. Rapid and sensitive detection of citrus bacterial canker by loop-mediated isothermal amplification combined with simple visual evaluation methods. BMC Microbiol. 2010;10:176.  https://doi.org/10.1186/1471-2180-10-176.CrossRefGoogle Scholar
  16. 16.
    Ge Y, Wu B, Qi X, Zhao K, Guo X, Zhu Y, et al. Rapid and sensitive detection of novel avian-origin influenza A (H7N9) virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device. PLoS One. 2013;8(8):e69941.  https://doi.org/10.1371/journal.pone.0069941.CrossRefGoogle Scholar
  17. 17.
    Roskos K, Hickerson AI, Lu HW, Ferguson TM, Shinde DN, Klaue Y, et al. Simple system for isothermal DNA amplification coupled to lateral flow detection. PLoS One. 2013;8(7):e69355.  https://doi.org/10.1371/journal.pone.0069355.CrossRefGoogle Scholar
  18. 18.
    Nurul Najian AB, Engku Nur Syafirah EA, Ismail N, Mohamed M, Yean CY. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira. Anal Chim Acta. 2016;903:142–8.  https://doi.org/10.1016/j.aca.2015.11.015.CrossRefGoogle Scholar
  19. 19.
    Kaewphinit T, Arunrut N, Kiatpathomchai W, Santiwatanakul S, Jaratsing P, Chansiri K. Detection of Mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples. Biomed Res Int. 2013;2013:926230.  https://doi.org/10.1155/2013/926230.CrossRefGoogle Scholar
  20. 20.
    Rigano LA, Malamud F, Orce IG, Filippone MP, Marano MR, do Amaral AM, et al. Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick. BMC Microbiol. 2014;14:86.  https://doi.org/10.1186/1471-2180-14-86.CrossRefGoogle Scholar
  21. 21.
    Prompamorn P, Sithigorngul P, Rukpratanporn S, Longyant S, Sridulyakul P, Chaivisuthangkura P. The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus. Lett Appl Microbiol. 2011;52(4):344–51.  https://doi.org/10.1111/j.1472-765X.2011.03007.x.CrossRefGoogle Scholar
  22. 22.
    Sun YL, Yen CH, Tu CF. Visual detection of canine parvovirus based on loop-mediated isothermal amplification combined with enzyme-linked immunosorbent assay and with lateral flow dipstick. J Vet Med Sci. 2014;76(4):509–16.CrossRefGoogle Scholar
  23. 23.
    Singh DV, Mohapatra H. Application of DNA-based methods in typing Vibrio cholerae strains. Future Microbiol. 2008;3(1):87–96.  https://doi.org/10.2217/17460913.3.1.87.CrossRefGoogle Scholar
  24. 24.
    Faruque SM, Kamruzzaman M, Meraj IM, Chowdhury N, Nair GB, Sack R, et al. Pathogenic potential of environmental Vibrio cholerae strains carrying genetic variants of the toxin-coregulated pilus pathogenicity island. Infect Immun. 2003;71(2):1020–5.CrossRefGoogle Scholar
  25. 25.
    Boyer SL, Flechtner VR, Johansen JR. Is the 16S-23S rRNA internal transcribed spacer region a good tool for use in molecular systematics and population genetics? A case study in cyanobacteria. Mol Biol Evol. 2001;18(6):1057–69.  https://doi.org/10.1093/oxfordjournals.molbev.a003877.CrossRefGoogle Scholar
  26. 26.
    Chun J, Huq A, Colwell RR. Analysis of 16S-23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus. Appl Environ Microbiol. 1999;65(5):2202–8.Google Scholar
  27. 27.
    ISO. Microbiology of the food chain - horizontal method for the determination of Vibrio spp. - part 1: detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. 2017.Google Scholar
  28. 28.
    Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002;16(3):223–9.CrossRefGoogle Scholar
  29. 29.
    Bashir A, Klammer A, Robins WP, Chin CS, Webster D, Paxinos E, et al. A hybrid approach for the automated finishing of bacterial genomes. Nat Biotechnol. 2012;30(7):701–7.  https://doi.org/10.1038/nbt.2288.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Life SciencesQingdao UniversityQingdaoChina
  2. 2.School of MedicineNankai UniversityTianjinChina
  3. 3.Tianjin International Travel Health Care CenterTianjinChina

Personalised recommendations