Skip to main content

Advertisement

Log in

A Real-Time PCR with Melting Curve Analysis for Molecular Typing of Vibrio parahaemolyticus

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

Foodborne disease caused by Vibrio parahaemolyticus is a serious public health problem in many countries. Molecular typing has a great scientific significance and application value for epidemiological research of V. parahaemolyticus. In this study, a real-time PCR with melting curve analysis was established for molecular typing of V. parahaemolyticus. Eighteen large variably presented gene clusters (LVPCs) of V. parahaemolyticus which have different distributions in the genome of different strains were selected as targets. Primer pairs of 18 LVPCs were distributed into three tubes. To validate this newly developed assay, we tested 53 Vibrio parahaemolyticus strains, which were classified in 13 different types. Furthermore, cluster analysis using NTSYS PC 2.02 software could divide 53 V. parahaemolyticus strains into six clusters at a relative similarity coefficient of 0.85. This method is fast, simple, and conveniently for molecular typing of V. parahaemolyticus.

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

Access this article

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Wang S, Duan H, Zhang W, Li JW (2007) Analysis of bacterial foodborne disease outbreaks in China between 1994 and 2005. FEMS Immunol Med Microbiol 51:8–13. https://doi.org/10.1111/j.1574-695X.2007.00305.x

    Article  PubMed  CAS  Google Scholar 

  2. Iwamoto M, Ayers T, Mahon BE, Swerdlow DL (2010) Epidemiology of seafood-associated infections in the United States. Clin Microbiol Rev 23:399–411. https://doi.org/10.1128/CMR.00059-09

    Article  PubMed  PubMed Central  Google Scholar 

  3. Mahmud ZH, Neogi SB, Kassu A, Wada T, Islam MS, Nair GB, Ota F (2007) Seaweeds as a reservoir for diverse Vibrio parahaemolyticus populations in Japan. Int J Food Microbiol 118:92–96. https://doi.org/10.1016/j.ijfoodmicro.2007.05.009

    Article  PubMed  Google Scholar 

  4. Lüdeke CH, Gonzalez-Escalona N, Fischer M, Jones JL (2015) Examination of clinical and environmental Vibrio parahaemolyticus isolates by multi-locus sequence typing (MLST) and multiple-locus variable-number tandem-repeat analysis (MLVA). Front Microbiol 6:564. https://doi.org/10.3389/fmicb.2015.00564

    Article  PubMed  PubMed Central  Google Scholar 

  5. Lüdeke CH, Fischer M, LaFon P, Cooper K, Jones JL (2014) Suitability of the molecular subtyping methods intergenic spacer region, direct genome restriction analysis, and pulsed-field gel electrophoresis for clinical and environmental Vibrio parahaemolyticus isolates. Foodborne Pathog Dis 11:520–528. https://doi.org/10.1089/fpd.2013.1728

    Article  PubMed  CAS  Google Scholar 

  6. Sahilah AM, Laila RA, Sallehuddin HM, Osman H, Aminah A, Ahmad Azuhairi A (2014) Antibiotic resistance and molecular typing among cockle (Anadara granosa) strains of Vibrio parahaemolyticus by polymerase chain reaction (PCR)-based analysis. World J Microbiol Biotechnol 30:649–659. https://doi.org/10.1007/s11274-013-1494-y

    Article  PubMed  CAS  Google Scholar 

  7. Heo EJ, Song BR, Park HJ, Kim YJ, Moon JS, Wee SH, Kim JS, Yoon Y (2014) Rapid detection of Listeria monocytogenes by real-time PCR in processed meat and dairy products. J Food Prot 77:453–458. https://doi.org/10.4315/0362-028X.JFP-13-318

    Article  PubMed  Google Scholar 

  8. Botaro BG, Cortinhas CS, Março LV, Moreno JF, Silva LF, Benites NR, Santos MV (2013) Detection and enumeration of Staphylococcus aureus from bovine milk samples by real-time polymerase chain reaction. J Dairy Sci 96:6955–6964

    Article  PubMed  CAS  Google Scholar 

  9. Liu B, He X, Chen W, Yu S, Shi C, Zhou X, Chen J, Wang D, Shi X (2012) Development of a real-time PCR assay for rapid detection of Vibrio parahaemolyticus from seafood. Protein Cell 3:204–212. https://doi.org/10.1007/s13238-012-2017-6

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Okada N, Iida T, Park KS, Goto N, Yasunaga T, Hiyoshi H, Matsuda S, Kodama T, Honda T (2009) Identification and characterization of a novel type III secretion system in trh-positive Vibrio parahaemolyticus strain TH3996 reveal genetic lineage and diversity of pathogenic machinery beyond the species level. Infect Immun 77:904–913. https://doi.org/10.1128/IAI.01184-08

    Article  PubMed  CAS  Google Scholar 

  11. Izutsu K, Kurokawa K, Tashiro K, Kuhara S, Hayashi T, Honda T, Iida T (2008) Comparative genomic analysis using microarray demonstrates a strong correlation between the presence of the 80-kilobase pathogenicity island and pathogenicity in Kanagawa phenomenon-positive Vibrio parahaemolyticus strains. Infect Immun 76:1016–1023. https://doi.org/10.1128/IAI.01535-07

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Xiao X, Yan Y, Zhang Y, Wang L, Liu X, Yang L, Tan Y, Guo Z, Yang R, Zhou D (2011) A novel genotyping scheme for Vibrio parahaemolyticus with combined use of large variably-presented gene clusters (LVPCs) and variable-number tandem repeats (VNTRs). Int J Food Microbiol 149:143–151. https://doi.org/10.1016/j.ijfoodmicro.2011.06.014

    Article  PubMed  CAS  Google Scholar 

  13. Mala W, Alam M, Angkititrakul S, Wongwajana S, Lulitanond V, Huttayananont S, Kaewkes W, Faksri K, Chomvarin C (2016) Serogroup, virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand. Infect Genet Evol 39:212–218. https://doi.org/10.1016/j.meegid.2016.01.006

    Article  PubMed  CAS  Google Scholar 

  14. Tsai SE, Jong KJ, Tey YH, Yu WT, Chiou CS, Lee YS, Wong HC (2013) Molecular characterization of clinical and environmental Vibrio parahaemolyticus isolates in Taiwan. Int J Food Microbiol 165:18–26. https://doi.org/10.1016/j.ijfoodmicro.2013.04.017

    Article  PubMed  CAS  Google Scholar 

  15. Chowdhury G, Ghosh S, Pazhani GP, Paul BK, Maji D, Mukhopadhyay AK, Ramamurthy T (2013) Isolation and characterization of pandemic and nonpandemic strains of Vibrio parahaemolyticus from an outbreak of diarrhea in North 24 Parganas, West Bengal, India. Foodborne Pathog Dis 10:338–342. https://doi.org/10.1089/fpd.2012.1340

    Article  PubMed  CAS  Google Scholar 

  16. He P, Chen Z, Luo J, Wang H, Yan Y, Chen L, Gao W (2014) Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains. Mol Cell Probes 28:246–250. https://doi.org/10.1016/j.mcp.2014.06.001

    Article  PubMed  CAS  Google Scholar 

  17. Robert-Pillot A, Copin S, Gay M, Malle P, Quilici ML (2010) Total and pathogenic Vibrio parahaemolyticus in shrimp: fast and reliable quantification by real-time PCR. Int J Food Microbiol 143:190–197. https://doi.org/10.1016/j.ijfoodmicro.2010.08.016

    Article  PubMed  CAS  Google Scholar 

  18. Nordstrom JL, Vickery MC, Blackstone GM, Murray SL, De Paola A (2007) Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters. Appl Environ Microbiol 73:5840–5847. https://doi.org/10.1128/AEM.00460-07

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Pang Y, Zhou Y, Wang S, Lu J, Lu B, He G, Wang L, Zhao Y (2011) A novel method based on high resolution melting (HRM) analysis for MIRU-VNTR genotyping of Mycobacterium tuberculosis. J Microbiol Methods 86:291–297. https://doi.org/10.1016/j.mimet.2011.05.016

    Article  PubMed  CAS  Google Scholar 

  20. Tong SY, Giffard PM (2012) Microbiological applications of high-resolution melting analysis. J Clin Microbiol 50:3418–3421. https://doi.org/10.1128/JCM.01709-12

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Han H, Wong HC, Kan B, Guo Z, Zeng X, Yin S, Liu X, Yang R, Zhou D (2008) Genome plasticity of Vibrio parahaemolyticus: microevolution of the ‘pandemic group’. BMC Genom 9:570. https://doi.org/10.1186/1471-2164-9-570

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by Science and Technology Program of Jiaxing City (No. 2013AY21051-1 and 2017AY33071).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Zhongwen Chen.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1: Online Resource 1 (PDF 210 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

He, P., Wang, H., Luo, J. et al. A Real-Time PCR with Melting Curve Analysis for Molecular Typing of Vibrio parahaemolyticus. Curr Microbiol 75, 1206–1213 (2018). https://doi.org/10.1007/s00284-018-1511-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-018-1511-3

Navigation