Rapid detection of  Clostridium perfringens  in food by loop-mediated isothermal amplification combined with a lateral flow biosensor

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Abstract

Clostridium perfringens is a key anaerobic pathogen causing food poisoning. Definitive detection by standard culture method is time-consuming and labor intensive. Current rapid commercial test kits are prohibitively expensive. It is thus necessary to develop rapid and cost-effective detection tool. Here, loop-mediated isothermal amplification (LAMP) in combination with a lateral-flow biosensor (LFB) was developed for visual inspection of C. perfringens-specific cpa gene. The specificity of the developed test was evaluated against 40 C. perfringens and 35 other bacterial strains, which showed no cross-reactivity, indicating 100% inclusivity and exclusivity. LAMP-LFB detection limit for artificially contaminated samples after enrichment for 16 h was 1–10 CFU/g sample, which was comparable to the commercial real-time PCR kit. The detection performance of LAMP-LFB was also compared to culture-based method using 95 food samples, which revealed the sensitivity (SE), specificity (SP) and Cohen's kappa coefficient (κ) of 88.0% (95% CI, 75.6%-95.4%), 95.5% (95% CI, 84.8%-99.4%) and 0.832 (95% CI, 0.721–0.943), respectively. Area under the receiver operating characteristic (ROC) curve was 0.918 (95% CI, 0.854–0.981), indicating LAMP-LFB as high relative accuracy test. In conclusion, LAMP-LFB assay is a low-cost qualitative method and easily available for routine detection of C. perfringens in food samples, which could serve as an alternative to commercial test kit.

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Most cited references 27

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Loop-mediated isothermal amplification of DNA.

T. Notomi (2000)

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

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Clostridial enteric diseases of domestic animals.

J Songer (1996)

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Loop‐mediated isothermal amplification (LAMP): a versatile technique for detection of micro‐organisms

Y-P Wong, S. Othman, Y.-L. Lau … (2018)

Summary Loop‐mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. (2000 Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro‐organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro‐organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real‐time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro‐organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease.

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Author and article information

Contributors

Thanawat Sridapan: Role: Data curationRole: Formal analysisRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draft

Wanida Tangkawsakul: Role: Methodology

Tavan Janvilisri: Role: Funding acquisitionRole: Writing – review & editing

Wansika Kiatpathomchai: Role: Funding acquisitionRole: Resources

Sirintip Dangtip: Role: Resources

Natharin Ngamwongsatit: Role: Resources

Duangjai Nacapricha: Role: Funding acquisition

Puey Ounjai:

ORCID: https://orcid.org/0000-0002-3335-6199

Role: Funding acquisition

Surang Chankhamhaengdecha:

ORCID: https://orcid.org/0000-0002-5414-4072

Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: ResourcesRole: SupervisionRole: Writing – review & editing

Yung-Fu Chang: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS One

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 7 January 2021

Publication date Collection: 2021

Volume: 16

Issue: 1

Electronic Location Identifier: e0245144

Affiliations

[1 ] Graduate Program in Molecular Medicine, Faculty of Science, Mahidol University, Bangkok, Thailand

[2 ] Center of Nanoscience and Nanotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand

[3 ] Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand

[4 ] Bioengineering and Sensing Technology Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand

[5 ] Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand

[6 ] Department of Chemistry, Faculty of Science, Mahidol University, Bangkok, Thailand

[7 ] Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand

Cornell University, UNITED STATES

Author notes

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: surang.cha@ 123456mahidol.ac.th

Author information

Puey Ounjai https://orcid.org/0000-0002-3335-6199

Surang Chankhamhaengdecha https://orcid.org/0000-0002-5414-4072

Article

Publisher ID: PONE-D-20-30204

DOI: 10.1371/journal.pone.0245144

PMC ID: 7790239

PubMed ID: 33411848

SO-VID: ccabefe1-062c-473c-a305-fe2efbbe09a7

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 24 September 2020

Date accepted : 22 December 2020