Skip to main content
SpringerLink
Log in

Real-time Loop-Mediated Isothermal Amplification (LAMP) Using Self-quenching Fluorogenic Probes: the Application in Skipjack Tuna (Katsuwonus pelamis) Authentication

  • Published:
Food Analytical Methods Aims and scope Submit manuscript

Abstract

Target-specific detection of LAMP amplicons can be achieved through the use of target-specific probes or modified primers as biorecognition elements, decreasing the likelihood of detecting false-positive signals. The present work selected skipjack tuna as a case study, and developed a novel LAMP assay coupled with the self-quenching fluorogenic probe for rapid detection of skipjack tuna. Specifically, the inner forward primer (FIP) was identified as the candidate primer to design the self-quenching fluorogenic probe. The fluorophore attached to the FIP (FIP-FAM) is self-quenched in unbound state. After binding to the dumbbell-shaped DNA target specifically, the FAM fluorophore is de-quenched, resulting in the fluorescence development. With the novel assay, as little as 5 fg of skipjack tuna, DNA could be detected. Moreover, the novel assay embraces a higher tolerance to impurities than non-specific fluorescent dyes, and succeeded the authentication of skipjack tuna from 6 commercial tuna products.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Armani A, Tinacci L, Xiong X, Castigliego L, Gianfaldoni G, Alessandra G (2015) Fish species identification in canned pet food by BLAST and forensically informative nucleotide sequencing (FINS) analysis of short fragments of the mitochondrial 16s ribosomal RNA gene (16S rRNA). Food Control 50:821–830

    Article  CAS  Google Scholar 

  • Becherer L, Borst N, Bakheit M, Frischmann S, Zengerle R, von Stetten F (2020) Loop-mediated isothermal amplification (LAMP)-review and classification of methods for sequence-specific detection. Anal Methods-UK 12:717–746

    Article  Google Scholar 

  • Christiansen H, Fournier N, Hellemans B, Volckaert FAM (2018) Seafood substitution and mislabeling in Brussels’ restaurants and canteens. Food Control 85:66–75

    Article  Google Scholar 

  • Chuang P, Chen M, Shiao J (2012) Identification of tuna species by a real-time polymerase chain reaction technique. Food Chem 133:1055–1061

    Article  CAS  Google Scholar 

  • Dalmasso A, Fontanella E, Piatti P, Civera T, Secchi C, Bottero MT (2007) Identification of four tuna species by means of real-time PCR and melting curve analysis. Vet Res Commun 31(Suppl 1):355–357

    Article  Google Scholar 

  • FAO (2020) Fishery Statistical Collections. http://www.fao.org/fishery/statistics/global-producti on/en (accessed 20 July 2021).

  • Frigerio J, Marchesi C, Magoni C, Saliu F, Ballabio D, Consonni V, Gorini T, Galli F, Labra P (2021) Application of DNA mini-barcoding and infrared spectroscopy for the authentication of the Italian product “bottarga”. LWT 139, 110603.

  • Gadkar VJ, Goldfarb DM, Gantt S, Tilley PAG (2018) Real-time detection and monitoring of loop mediated amplification (LAMP) reaction using self-quenching and de-quenching fluorogenic probes. SCI REP-UK 8.

  • Girish PS, Barbuddhe SB, Kumari A, Rawool DB, Karabasanavar NS, Muthukumar M, Vaithiyanathan S (2020) Rapid detection of pork using alkaline lysis- loop mediated isothermal amplification (AL-LAMP) technique. Food Control 110, 107015.

  • Hu Y, Huang SY, Hanner R, Levin J, Lu X (2018) Study of fish products in Metro Vancouver using DNA barcoding methods reveals fraudulent labeling. Food Control 94:38–47

    Article  CAS  Google Scholar 

  • Jiang YS, Bhadra S, Li BL, Wu YF, Milligan JN, Ellington AD (2015) Robust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons. Anal Chem 87(6):3314–3320

    Article  CAS  Google Scholar 

  • Kotsanopoulos KV, Exadactylos A, Gkafas GA, Martsikalis PV, Parlapani FF, Boziaris IS, Arvanitoyannis IS (2021) The use of molecular markers in the verification of fish and seafood authenticity and the detection of adulteration. Compr Rev Food Sci F 20:1584–1654

    Article  CAS  Google Scholar 

  • Lee D, Kim EJ, Kilgore PE, Kim SA, Takahashi H, Ohnishi M, Anh DD, Dong BQ, Kim JS, Tomono J, Miyamoto S, Notomi T, Kim DW, Seki M (2015) Clinical evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid. Plos One 10, e122922.

  • Liu W, Huang S, Liu N, Dong D, Yang Z, Tang Y, Ma W, He X, Xu Y, Zou D, Huang L (2017). Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay. SCI REP-UK-7.

  • Meagher RJ, Priye A, Light YK, Huang C, Wang E (2018) Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA. Analyst 143:1924–1933

    Article  CAS  Google Scholar 

  • Mitchell JK, Hellberg RS (2016) Use of the mitochondrial control region as a potential DNA mini-barcoding target for the identification of canned tuna species. Food Anal Method 9:2711–2720

    Article  Google Scholar 

  • Mohan CO, Remya S, Ravishankar CN, Vijayan PK, Srinivasa Gopal TK (2014) Effect of filling ingredient on the quality of canned yellowfin tuna (Thunnus albacares). Int J Food Sci Tech 49:1557–1564

    Article  CAS  Google Scholar 

  • Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16:223–229

    Article  CAS  Google Scholar 

  • Nazarenko I, Pires R, Lowe B, Obaidy M, Rashtchian A (2002) Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes. Nucleic Acids Res 30:2089–2195

    Article  CAS  Google Scholar 

  • Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28, e63.

  • Saull J, Duggan C, Hobbs G, Edwards T (2016) The detection of Atlantic cod (Gadus morhua) using loop mediated isothermal amplification in conjunction with a simplified DNA extraction process. Food Control 59:306–313

    Article  CAS  Google Scholar 

  • Servusova E, Piskata Z (2021) Identification of selected tuna species in commercial products. Molecules 26(4):1137

    Article  CAS  Google Scholar 

  • Sotelo CG, Velasco A, Perez-Martin RI, Kappel K, Schröder U, Verrez-Bagnis V, Jérôme M, Mendes R, Silva H, Mariani S, Griffiths A (2018) Tuna labels matter in Europe: mislabelling rates in different tuna products. Plos One 13, e196641.

  • Spielmann G, Ziegler S, Haszprunar G, Busch U, Huber I, Pavlovic M (2019) Using loop-mediated isothermal amplification for fast species delimitation in eels (genus Anguilla), with special reference to the European eel (Anguilla anguilla). Food Control 101:156–162

    Article  CAS  Google Scholar 

  • Spink JW (2019) The current state of food fraud prevention: overview and requirements to address ‘How to Start?’ and ‘How Much is Enough?’ Curr Opin Food Sci 27:130–138

    Article  Google Scholar 

  • Vandamme SG, Griffiths AM, Taylor S, Di Muri C, Hankard EA, Towne JA, Watson M, Mariani S, Mikheyev A (2016) Sushi barcoding in the UK: another kettle of fish. Peerj 4, e1891.

  • Wang D, Brewster J, Paul M, Tomasula P (2015) Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification. Molecules 20:6048–6059

    Article  CAS  Google Scholar 

  • Xiong X, D’Amico P, Guardone L, Castigliego L, Guidi A, Gianfaldoni D, Armani A (2016) The uncertainty of seafood labeling in China: a case study on Cod. Salmon and Tuna Mar Policy 68:123–135

    Article  Google Scholar 

  • Xiong X, Huang M, Xu W, Li Y, Cao M, Xiong X (2021a) Using real time fluorescence loop-mediated isothermal amplification for rapid species authentication of Atlantic salmon (Salmo salar). J Food Compos Anal 95, 103659.

  • Xiong X, Xu W, Guo L, An J, Huang L, Qian H, Cui X, Li Y, Cao M, Xiong X, Ying X, Wang L (2021b) Development of loop-mediated isothermal amplification (LAMP) assay for rapid screening of skipjack tuna (Katsuwonus pelamis) in processed fish products. J Food Compos Anal 104038.

  • Yao L, Lu J, Qu M, Jiang Y, Li F, Guo Y, Wang L, Zhai Y (2020) Methodology and application of PCR-RFLP for species identification in tuna sashimi. Food Sci Nutr 8:3138–3146

    Article  CAS  Google Scholar 

  • Yoon IS, Lee G, Kang SI, Park SY, Lee JS, Kim J, Heu MS (2018) Chemical composition and functional properties of roe concentrates from skipjack tuna (Katsuwonus pelamis) by cook-dried process. Food Sci Nutr 6:1276–1286

    Article  CAS  Google Scholar 

  • Zhang M, Ye J, He J, Zhang F, Ping J, Qian C, Wu J (2020) Visual detection for nucleic acid-based techniques as potential on-site detection methods. A Review Anal Chim Acta 1099:1–15

    Article  CAS  Google Scholar 

Download references

Funding

This work was supported by the National Key Research and Development Program of China (No. 2018YFC1602800) and the National Natural Science Foundation of China (grant number 31701688).

Ethical Approval.

This article does not contain any studies with human or animals subjects.

Author information

Authors and Affiliations

  1. College of Food Science and Light Industry, Nanjing Tech University, No. 30 Puzhu Road South, Nanjing, 211800, China

    Wenjie Xu, Qiuping Li, Xiaowen Cui, Min Cao, Xiaohui Xiong & Xiong Xiong

  2. College of Light Industry and Food Engineering, Nanjing Forestry University, Jiangsu Province, Nanjing, 210037, China

    Libin Wang

Authors
  1. Wenjie Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qiuping Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaowen Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Min Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaohui Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Libin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiong Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Investigation, methodology, writing (original draft), Xu Wenjie; resources, investigation, Li Qiuping; methodology, Cui Xiaowen; visualization, Cao Min; writing (review and editing), Xiong Xiaohui; writing (review and editing), Wang Libin; data curation, writing (review and editing), project administration, Xiong Xiong.

Corresponding author

Correspondence to Xiong Xiong.

Ethics declarations

Consent to Participate

All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

Xu Wenjie declares that he has no conflict of interest. Li Qiuping declares that she has no conflict of interest. Cui Xiaowen declares that he has no conflict of interest. Min Cao declares that she has no conflict of interest. Xiaohui Xiong declares that he has no conflict of interest. Wang Libin declares that he has no conflict of interest. Xiong Xiong declares that he has no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, W., Li, Q., Cui, X. et al. Real-time Loop-Mediated Isothermal Amplification (LAMP) Using Self-quenching Fluorogenic Probes: the Application in Skipjack Tuna (Katsuwonus pelamis) Authentication. Food Anal. Methods 15, 658–665 (2022). https://doi.org/10.1007/s12161-021-02159-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12161-021-02159-1

Keywords

Search

Navigation