Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V. parahaemolyticus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Culture Medium, and Spiked Sample Preparation
2.2. DNA Extraction
2.3. PCR and PCR-LFD Assay
2.4. Specificity and Sensitivity Testing
2.5. Artificial Spiking Experiment
3. Results
3.1. Optimization of Multiplex PCR-LFD Assay
3.2. Specificity Evaluation of Multiplex PCR-LFD Assay
3.3. Sensitivity Determination of Multiplex PCR-LFD Assay
3.4. Detection of Pathogenic TDH+ V. parahaemolyticus-Contaminated Samples Using Multiplex PCR-LFD Assay
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Broberg, C.A.; Calder, T.J.; Orth, K.V. parahaemolyticus Cell Biology and Pathogenicity Determinants. Microbes Infect. 2011, 13, 992–1001. [Google Scholar] [CrossRef] [Green Version]
- Yeung, P.S.M.; Boor, K.J. Epidemiology, Pathogenesis, and Prevention of Foodborne V. parahaemolyticus Infections. Foodborne Pathog. Dis. 2004, 1, 74–88. [Google Scholar] [CrossRef]
- Ellett, A.N.; Rosales, D.; Jacobs, J.M.; Paranjpye, R.; Parveen, S. Growth Rates of V. parahaemolyticus Sequence Type 36 Strains in Live Oysters and in Culture Medium. Microbiol. Spectr. 2022, 10, e02112-22. [Google Scholar] [CrossRef]
- Baker-Austin, C.; Trinanes, J.; Gonzalez-Escalona, N.; Martinez-Urtaza, J. Non-Cholera Vibrios: The Microbial Barometer of Climate Change. Trends Microbiol. 2017, 25, 76–84. [Google Scholar] [CrossRef] [Green Version]
- Wang, R.; Zhong, Y.; Gu, X.; Yuan, J.; Saeed, A.F.; Wang, S. The Pathogenesis, Detection, and Prevention of V. parahaemolyticus. Front. Microbiol. 2015, 6, 144. [Google Scholar] [CrossRef] [PubMed]
- Li, L.; Meng, H.; Gu, D.; Li, Y.; Jia, M. Molecular Mechanisms of V. parahaemolyticus Pathogenesis. Microbiol. Res. 2019, 222, 43–51. [Google Scholar] [CrossRef] [PubMed]
- Theethakaew, C.; Feil, E.J.; Castillo-Ramírez, S.; Aanensen, D.M.; Suthienkul, O.; Neil, D.M.; Davies, R.L. Genetic Relationships of V. parahaemolyticus Isolates from Clinical, Human Carrier, and Environmental Sources in Thailand, Determined by Multilocus Sequence Analysis. Appl. Environ. Microbiol. 2013, 79, 2358–2370. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yingkajorn, M.; Mitraparp-arthorn, P.; Nuanualsuwan, S.; Poomwised, R.; Kongchuay, N.; Khamhaeng, N.; Vuddhakul, V. Prevalence and Quantification of Pathogenic V. parahaemolyticus during Shrimp Culture in Thailand. Dis. Aquat. Org. 2014, 112, 103–111. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Thongjun, J.; Mittraparp-arthorn, P.; Yingkajorn, M.; Kongreung, J.; Nishibuchi, M.; Vuddhakul, V. The Trend of V. parahaemolyticus Infections in Southern Thailand from 2006 to 2010. Trop. Med. Health 2013, 41, 151–156. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tran, T.H.T.; Yanagawa, H.; Nguyen, K.T.; Hara-Kudo, Y.; Taniguchi, T.; Hayashidani, H. Prevalence of V. parahaemolyticus in Seafood and Water Environment in the Mekong Delta, Vietnam. J. Vet. Med. Sci. 2018, 80, 1737–1742. [Google Scholar] [CrossRef] [Green Version]
- Raghunath, P. Roles of Thermostable Direct Hemolysin (TDH) and TDH-Related Hemolysin (TRH) in V. parahaemolyticus. Front. Microbiol. 2014, 5, 805. [Google Scholar] [CrossRef] [PubMed]
- Kulkarni, M.B.; Ayachit, N.H.; Aminabhavi, T.M. Recent Advances in Microfluidics-Based Electrochemical Sensors for Foodborne Pathogen Detection. Biosensors 2023, 9, 246. [Google Scholar] [CrossRef] [PubMed]
- Janik-Karpinska, E.; Ceremuga, M.; Niemcewicz, M.; Podogrocki, M.; Stela, M.; Cichon, N.; Bijak, M. Immunosensors—The Future of Pathogen Real-Time Detection. Sensors 2022, 22, 9757. [Google Scholar] [CrossRef] [PubMed]
- Federici, S.; Serrazanetti, D.I.; Guerzoni, M.E.; Campana, R.; Ciandrini, E.; Baffone, W.; Gianotti, A. Development of a Rapid PCR Protocol to Detect V. parahaemolyticus in Clams. J. Food Sci. Technol. 2018, 55, 749–759. [Google Scholar] [CrossRef]
- Park, J.Y.; Jeon, S.; Kim, J.Y.; Park, M.; Kim, S. Multiplex Real-Time Polymerase Chain Reaction Assays for Simultaneous Detection of V. cholerae, V. parahaemolyticus, and V. vulnificus. Osong Public Health Res. Perspect. 2013, 4, 133–139. [Google Scholar] [CrossRef] [Green Version]
- Yu, J.; Xing, J.; Zhan, X.; Yang, Z.; Qi, J.; Wei, Y.; Liu, Y. Improvement of Loop-Mediated Isothermal Amplification Combined with Chromatographic Flow Dipstick Assay for Salmonella in Food Samples. Food Anal. Methods 2020, 13, 1398–1408. [Google Scholar] [CrossRef]
- Allgöwer, S.M.; Hartmann, C.A.; Holzhauser, T. The Development of Highly Specific and Sensitive Primers for the Detection of Potentially Allergenic Soybean (Glycine Max) Using Loop-Mediated Isothermal Amplification Combined with Lateral Flow Dipstick (LAMP-LFD). Foods 2020, 9, 423. [Google Scholar] [CrossRef] [Green Version]
- Dai, T.; Yang, X.; Hu, T.; Jiao, B.; Xu, Y.; Zheng, X.; Shen, D. Comparative Evaluation of a Novel Recombinase Polymerase Amplification-Lateral Flow Dipstick (RPA-LFD) Assay, LAMP, Conventional PCR, and Leaf-Disc Baiting Methods for Detection of Phytophthora sojae. Front. Microbiol. 2019, 10, 1884. [Google Scholar] [CrossRef] [Green Version]
- Bej, A.K.; Patterson, D.P.; Brasher, C.W.; Vickery, M.C.; Jones, D.D.; Kaysner, C.A. Detection of Total and Hemolysin-Producing V. parahaemolyticus in Shellfish Using Multiplex PCR Amplification of Tl, TDH and TRH. J. Microbiol. Methods 1999, 36, 215–225. [Google Scholar] [CrossRef]
- World Health Organization; Food and Agriculture Organization of the United Nations. Risk Assessment of V. parahaemolyticus in Seafood: Interpretative Summary and Technical Report; World Health Organization: Geneva, Switzerland, 2011.
- Wang, P.; Liao, L.; Ma, C.; Zhang, X.; Yu, J.; Yi, L.; Liu, X.; Shen, H.; Gao, S.; Lu, Q. Duplex On-Site Detection of V. cholerae and V. vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips. Biosensors 2021, 11, 151. [Google Scholar] [CrossRef]
- Palamae, S.; Mittal, A.; Yingkajorn, M.; Saetang, J.; Buatong, J.; Tyagi, A.; Singh, P.; Benjakul, S.V. parahaemolyticus Isolates from Asian Green Mussel: Molecular Characteristics, Virulence and Their Inhibition by Chitooligosaccharide-Tea Polyphenol Conjugates. Foods 2022, 11, 4048. [Google Scholar] [CrossRef] [PubMed]
- Kaysner, C.A.; DePaola, A., Jr.; Jones, J. BAM Chapter 9: Vibrio; FDA: Rome, Italy, 2020.
- Kang, C.-H.; Shin, Y.; Kim, W.; Kim, Y.; Song, K.; Oh, E.-G.; Kim, S.; Yu, H.; So, J.-S. Prevalence and Antimicrobial Susceptibility of V. parahaemolyticus Isolated from Oysters in Korea. Environ. Sci. Pollut. Res. Int. 2016, 23, 918–926. [Google Scholar] [CrossRef] [PubMed]
- Law, J.W.-F.; Ab Mutalib, N.-S.; Chan, K.-G.; Lee, L.-H. Rapid Methods for the Detection of Foodborne Bacterial Pathogens: Principles, Applications, Advantages and Limitations. Front. Microbiol. 2014, 5, 770. [Google Scholar] [CrossRef] [Green Version]
- Gavilan, R.G.; Caro-Castro, J.; Blondel, C.J.; Martinez-Urtaza, J. Vibrio parahaemolyticus Epidemiology and Pathogenesis: Novel Insights on an Emerging Foodborne Pathogen. Adv. Exp. Med. Biol. 2023, 1404, 233–251. [Google Scholar] [CrossRef]
- Hariri, S. Detection of Escherichia coli in Food Samples Using Culture and Polymerase Chain Reaction Methods. Cureus 2022, 14, e32808. [Google Scholar] [CrossRef]
- Awang, M.S.; Bustami, Y.; Hamzah, H.H.; Zambry, N.S.; Najib, M.A.; Khalid, M.F.; Aziah, I.; Abd Manaf, A. Advancement in Salmonella Detection Methods: From Conventional to Electrochemical-Based Sensing Detection. Biosensors 2021, 11, 346. [Google Scholar] [CrossRef]
- Brengi, S.P.; Sun, Q.; Bolaños, H.; Duarte, F.; Jenkins, C.; Pichel, M.; Shahnaij, M.; Sowers, E.G.; Strockbine, N.; Talukder, K.A.; et al. PCR-Based Method for Shigella flexneri Serotyping: International Multicenter Validation. J. Clin. Microbiol. 2019, 57, e01592-18. [Google Scholar] [CrossRef] [Green Version]
- Molina, F.; López-Acedo, E.; Tabla, R.; Roa, I.; Gómez, A.; Rebollo, J.E. Improved Detection of Escherichia coli and Coliform Bacteria by Multiplex PCR. BMC Biotechnol. 2015, 15, 48. [Google Scholar] [CrossRef] [Green Version]
- Chin, W.H.; Sun, Y.; Høgberg, J.; Quyen, T.L.; Engelsmann, P.; Wolff, A.; Bang, D.D. Direct PCR—A Rapid Method for Multiplexed Detection of Different Serotypes of Salmonella in Enriched Pork Meat Samples. Mol. Cell. Probes 2017, 32, 24–32. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhang, M.; Wu, J.; Shi, Z.; Cao, A.; Fang, W.; Yan, D.; Wang, Q.; Li, Y. Molecular Methods for Identification and Quantification of Foodborne Pathogens. Molecules 2022, 27, 8262. [Google Scholar] [CrossRef]
- Mukhopadhyay, A.; Mukhopadhyay, U.K. Novel Multiplex PCR Approaches for the Simultaneous Detection of Human Pathogens: Escherichia coli 0157:H7 and Listeria monocytogenes. J. Microbiol. Methods 2007, 68, 193–200. [Google Scholar] [CrossRef] [PubMed]
- Phuakrod, A.; Sripumkhai, W.; Jeamsaksiri, W.; Pattamang, P.; Loymek, S.; Brindley, P.J.; Sarasombath, P.T.; Wongkamchai, S. A MiniPCR-Duplex Lateral Flow Dipstick Platform for Rapid and Visual Diagnosis of Lymphatic Filariae Infection. Diagnostics 2021, 11, 1855. [Google Scholar] [CrossRef] [PubMed]
- Yin, R.; Sun, Y.; Wang, K.; Feng, N.; Zhang, H.; Xiao, M. Development of a PCR-Based Lateral Flow Strip Assay for the Simple, Rapid, and Accurate Detection of Pork in Meat and Meat Products. Food Chem. 2020, 318, 126541. [Google Scholar] [CrossRef] [PubMed]
- Taboada, L.; Sánchez, A.; Pérez-Martín, R.I.; Sotelo, C.G. A New Method for the Rapid Detection of Atlantic Cod (Gadus morhua), Pacific Cod (Gadus macrocephalus), Alaska Pollock (Gadus chalcogrammus) and Ling (Molva molva) Using a Lateral Flow Dipstick Assay. Food Chem. 2017, 233, 182–189. [Google Scholar] [CrossRef]
- Kwawukume, S.; Velez, F.J.; Williams, D.; Cui, L.; Singh, P. Rapid PCR-Lateral Flow Assay for the Onsite Detection of Atlantic White Shrimp. Food Chem. 2023, 6, 100164. [Google Scholar] [CrossRef]
- Xing, J.; Yu, J.; Liu, Y. Improvement and Evaluation of Loop-Mediated Isothermal Amplification Combined with Chromatographic Flow Dipstick Assays for V. parahaemolyticus. J. Microbiol. Methods 2020, 171, 105866. [Google Scholar] [CrossRef]
- Nordstrom, J.L.; Vickery, M.C.L.; Blackstone, G.M.; Murray, S.L.; DePaola, A. Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic V. parahaemolyticus Bacteria in Oysters. Appl. Environ. Microbiol. 2007, 73, 5840–5847. [Google Scholar] [CrossRef] [Green Version]
- Letchumanan, V.; Chan, K.-G.; Lee, L.-H. V. parahaemolyticus: A Review on the Pathogenesis, Prevalence, and Advance Molecular Identification Techniques. Front. Microbiol. 2014, 5, 705. [Google Scholar] [CrossRef] [Green Version]
- Hu, Y.; Huang, X.; Guo, L.; Shen, Z.; LV, L.; Li, F.; Zhou, Z.; Zhang, D. Rapid and Visual Detection of V. parahaemolyticus in Aquatic Foods Using BlaCARB-17 Gene-Based Loop-Mediated Isothermal Amplification with Lateral Flow Dipstick (LAMP-LFD). J. Microbiol. Biotechnol. 2021, 31, 1672–1683. [Google Scholar] [CrossRef]
- Chen, X.; Zhu, Q.; Liu, Y.; Wang, R.; Xie, H.; Chen, J.; Cheng, Y.; Zhang, H.; Cao, L.; Chen, Y. Pathogenic Characteristics of and Variation in V. parahaemolyticus Isolated from Acute Diarrhoeal Patients in Southeastern China from 2013 to 2017. Infect. Drug Resist. 2020, 13, 1307–1318. [Google Scholar] [CrossRef]
- Chen, Y.; Chen, X.; Yu, F.; Wu, M.; Wang, R.; Zheng, S.; Han, D.; Yang, Q.; Kong, H.; Zhou, F.; et al. Serology, Virulence, Antimicrobial Susceptibility and Molecular Characteristics of Clinical V. parahaemolyticus Strains Circulating in Southeastern China from 2009 to 2013. Clin. Microbiol. Infect. 2016, 22, 258.e9-16. [Google Scholar] [CrossRef] [Green Version]
- Niu, B.; Hong, B.; Zhang, Z.; Mu, L.; Malakar, P.K.; Liu, H.; Pan, Y.; Zhao, Y. A Novel QPCR Method for Simultaneous Detection and Quantification of Viable Pathogenic and Non-Pathogenic V. parahaemolyticus (Tlh+, TDH+, and UreR+). Front. Microbiol. 2018, 9, 1747. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hossain, M.T.; Kim, Y.-O.; Kong, I.-S. Multiplex PCR for the Detection and Differentiation of V. parahaemolyticus Strains Using the GroEL, TDH and TRH Genes. Mol. Cell. Probes 2013, 27, 171–175. [Google Scholar] [CrossRef]
- Ward, L.N.; Bej, A.K. Detection of V. parahaemolyticus in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes. Appl. Environ. Microbiol. 2006, 72, 2031–2042. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- He, P.; Chen, Z.; Luo, J.; Wang, H.; Yan, Y.; Chen, L.; Gao, W. Multiplex Real-Time PCR Assay for Detection of Pathogenic V. parahaemolyticus Strains. Mol. Cell. Probes 2014, 28, 246–250. [Google Scholar] [CrossRef]
- Wei, S.; Zhao, H.; Xian, Y.; Hussain, M.A.; Wu, X. Multiplex PCR Assays for the Detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae with an Internal Amplification Control. Diagn. Microbiol. Infect. Dis. 2014, 79, 115–118. [Google Scholar] [CrossRef] [PubMed]
- Sea-liang, N.; Sereemaspun, A.; Patarakul, K.; Gaywee, J.; Rodkvamtook, W.; Srisawat, N.; Wacharaplusadee, S.; Hemachudha, T. Development of Multiplex PCR for Neglected Infectious Diseases. PLoS Negl. Trop. Dis. 2019, 13, e0007440. [Google Scholar] [CrossRef] [PubMed]
- Lorenz, T.C. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. J. Vis. Exp. 2012, 63, 3998. [Google Scholar] [CrossRef]
- Sabat, G.; Rose, P.; Hickey, W.J.; Harkin, J.M. Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil. Appl. Environ. Microbiol. 2000, 66, 844–849. [Google Scholar] [CrossRef] [Green Version]
- Xu, D.; Ji, L.; Wu, X.; Yan, W.; Chen, L. Detection and Differentiation of V. parahaemolyticus by Multiplexed Real-Time PCR. Can. J. Microbiol. 2018, 64, 809–815. [Google Scholar] [CrossRef]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Saetang, J.; Sukkapat, P.; Palamae, S.; Singh, P.; Senathipathi, D.N.; Buatong, J.; Benjakul, S. Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V. parahaemolyticus. Biosensors 2023, 13, 698. https://doi.org/10.3390/bios13070698
Saetang J, Sukkapat P, Palamae S, Singh P, Senathipathi DN, Buatong J, Benjakul S. Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V. parahaemolyticus. Biosensors. 2023; 13(7):698. https://doi.org/10.3390/bios13070698
Chicago/Turabian StyleSaetang, Jirakrit, Phutthipong Sukkapat, Suriya Palamae, Prashant Singh, Deep Nithun Senathipathi, Jirayu Buatong, and Soottawat Benjakul. 2023. "Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V. parahaemolyticus" Biosensors 13, no. 7: 698. https://doi.org/10.3390/bios13070698