Year 2020, Volume 67 , Issue 4, Pages 349 - 355 2020-09-01

Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP

Yusuf DOĞRUER [1] , A. Ezgi TELLİ [2]


One of the most challenging aspects in culture independent methods for foodborne pathogens’ detection is discrimination of dead and live microorganisms. This study aimed to determine the Vibrio parahaemolyticus in seafoods via direct plate counting (DPC) and toxR-based quantitative loop-mediated isothermal amplification (qLAMP) and to discriminate dead and live cells using propidium monoazide (PMA)-qLAMP. A total of 200 samples including finfishes (n= 100) and shrimps (n= 100), representing the Mediterranean, Black and Aegean sea were collected from supermarkets and fish markets of Konya-Turkey. qLAMP was performed in a Real-Time Turbidimeter and the time threshold (tt) values were yielded in 60 minutes. On DPC, the colonies grown on TCBS Agar was further confirmed by conventional PCR based from gyrB1 gene of Vibrio spp. and toxR gene of V. parahaemolyticus. Virulence property of the isolates were determined by tdh based qLAMP. The detection limit of the qLAMP was 1.2×104 CFU/g in artificially contaminated seafoods. DPC, qLAMP and PMA-qLAMP detected V. parahaemolyticus in 8 (4%), 12 (6%) and 12 (6%) samples, respectively. The CFUs of V. parahaemolyticus detected in qLAMP (5.96±0.10 log10 CFU/ml) and PMA-qLAMP (4.71±0.13 log10 CFU/ml) were higher than those of DPC (1.99±0.44 log10 CFU/ml) (P<0.05). The mean tt reduction in PMA treated samples was 1.25±0.38 log10 CFU/sample. The tdh gene was not detected in any of the isolates. In conclusion, the toxR-based PMA-qLAMP method could be an alternative to be used more widely and effective assay for the quantification of live V. parahaemolyticus in seafoods.

DPC, propidium monoazide, qLAMP, VBNC, V. parahaemolyticus
  • 1. American Public Health Association (APHA) (1992): Standard Methods for the Examination of Water and Waste Water. American Public Health Association, Washington DC, USA.
  • 2. Austin B (2010): Vibrios as causal agents of zoonoses. Vet Microbiol, 140, 310-317.
  • 3. Bates TC, Oliver JD (2004): The viable but nonculturable state of Kanagawa positive and negative strains of Vibrio parahaemolyticus. J Microbiol, 42, 74-79.
  • 4. Cai T, Jiang L, Yang C, et al (2006): Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China. FEMS Immunol Med Microbiol, 46, 180-186.
  • 5. Chao G, Jiao X, Zhou X, et al (2009): Distribution, prevalence, molecular typing, and virulence of Vibrio parahaemolyticus isolated from different sources in coastal province Jiangsu, China. Food Control, 20, 907-912.
  • 6. Chen S, Ge B (2010): Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus. BMC Microbiol, 10, 41.
  • 7. Chen S, Wang F, Beaulieu JC, et al (2011): Rapid detection of viable Salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification (PMA-LAMP). Appl Environ Microbiol, 77, 4008-4016.
  • 8. DePaola A, Ulaszek J, Kaysner CA, et al (2003): Molecular, serological, and virulence characteristics of Vibrio parahaemolyticus isolated from environmental, food, and clinical sources in North America and Asia. Appl Environ Microbiol, 69, 3999-4005.
  • 9. Du M, Chen J, Zhang X, et al (2007): Retention of virulence in a viable but nonculturable Edwardsiella tarda isolate. Appl Environ Microbiol, 73, 1349-1354.
  • 10. Ducret A, Chabalier M, Dukan S (2014): Characterization and resuscitation of ‘non-culturable’cells of Legionella pneumophila. BMC Microbiol, 14, 3.
  • 11. Fang J, Wu Y, Qu D, et al (2018): Propidium monoazide real‐time loop‐mediated isothermal amplification for specific visualization of viable Salmonella in food. Lett Appl Microbiol, 67, 79-88.
  • 12. Farmer JJ (2005): Genus I. Vibrio pacini 1854. 494-546. In: GM Garrity (Ed), Bergey's Manual of Systematic Bacteriology. Springer, New York.
  • 13. García-Cayuela T, Tabasco R, Peláez C, et al (2009): Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR. Int Dairy J, 19, 405-409.
  • 14. Han F, Ge B (2010): Quantitative detection of Vibrio vulnificus in raw oysters by real-time loop-mediated isothermal amplification. Int J Food Microbiol, 142, 60-66.
  • 15. Jones JL, Hara-Kudo Y, Krantz, JA, et al (2012): Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus. Food Microbiol, 30, 105-111.
  • 16. Kokkinos PA, Ziros PG, Bellou M, et al (2014): Loop-mediated isothermal amplification (LAMP) for the detection of Salmonella in food. Food Anal Methods, 7, 512-526.
  • 17. Nagamine K, Hase T, Notomi T (2002): Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes, 16, 223-229.
  • 18. Nigro OD, Hou A, Vithanage G, et al (2011). Temporal and spatial variability in culturable pathogenic Vibrio spp. in lake Pontchartrain, Louisiana, following Hurricanes Katrina and Rita. Appl Environ Microbiol, 77, 5384-5393.
  • 19. Nocker A, Cheung CY, Camper AK (2006): Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods, 67, 310-320.
  • 20. Notomi T, Okayama H, Masubuchi H, et al (2000): Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 28, e63.
  • 21. Oberbeckmann S, Wichels A, Wiltshire KH, et al (2011): Occurrence of Vibrio parahaemolyticus and Vibrio alginolyticus in the German Bight over a seasonal cycle. Antonie Van Leeuwenhoek, 100, 291-307.
  • 22. Okada K, Chantaroj S, Taniguchi T, et al (2010): A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples. Diagn Microbiol Infect Dis, 66, 135-139.
  • 23. Oliver JD, Kaper JB (2007): Vibrio Species. 343-378. In: MP Doyle, LR Beuchat (Eds), Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington DC.
  • 24. Peterson KM (1999): Molecular pathogenesis of Vibrio infections. 157-190. In: JW Carry, JE Linz, D Bhatnagar (Eds), Microbial Foodborne Diseases: Mechanisms of Pathogenesis and Toxin Synthesis. Techomic Publishing, USA.
  • 25. Rawsthorne H, Dock CN, Jaykus LA (2009): PCR-based method using propidium monoazide to distinguish viable from nonviable Bacillus subtilis spores. Appl Environ Microbiol, 75, 2936-2939.
  • 26. Ren CH, Hu CQ, Luo P, et al (2009): Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification. Microbiol Res, 164, 514-521.
  • 27. Robert-Pillot A, Copin S, Himber C, et al (2014): Occurrence of the three major Vibrio species pathogenic for human in seafood products consumed in France using real-time PCR. Int J Food Microbiol, 189, 75-81.
  • 28. Rosec JP, Causse V, Cruz B, et al (2012): The international standard ISO/TS 21872–1 to study the occurence of total and pathogenic Vibrio parahaemolyticus and Vibrio cholerae in seafood: ITS improvement by use of a chromogenic medium and PCR. Int J Food Microbiol, 157, 189-194.
  • 29. Shanthini CF, Kumar PA, Patterson J (2004): Incidence and antibiotic susceptibility of Vibrio parahaemolyticus from sea foods of Tuticorin. Indian J Fish, 51, 43-47.
  • 30. Su YC, Liu C (2007): Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol, 24, 549-558.
  • 31. Sudha S, Divya PS, Francis B, et al (2012): Prevalence and distribution of Vibrio parahaemolyticus in finfish from Cochin (south India). Vet Ital, 48, 269-281.
  • 32. Techathuvanan C, Draughon FA, D'Souza DH (2011): Comparison of reverse transcriptase PCR, reverse transcriptase loop-mediated isothermal amplification, and culture-based assays for Salmonella detection from pork processing environments. J Food Prot, 74, 294-301.
  • 33. Teh CSJ, Chua KH, Thong KL (2010): Simultaneous differential detection of human pathogenic and nonpathogenic Vibrio species using a multiplex PCR based on gyrB and pntA genes. J Appl Microbiol, 108, 1940-1945.
  • 34. Telli AE, Dogruer Y (2019): Discrimination of viable and dead Vibrio parahaemolyticus subjected to low temperatures using propidium monoazide- quantitative loop mediated isothermal amplification (PMA-qLAMP) and PMA-qPCR. Microb Pathog, 132,109-116.
  • 35. Tomita N, Mori Y, Kanda H, et al (2008): Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc, 3, 877-882.
  • 36. Turkish Statistical Institue (2019): Fishery Statistics. Available at http://www.tuik.gov.tr/PreTablo.do?alt_id=1005. (Accessed March 30, 2019).
  • 37. Wan C, Yang Y, Xu H, et al (2012): Development of a propidium monoazide treatment combined with loop‐mediated isothermal amplification (PMA‐LAMP) assay for rapid detection of viable Listeria monocytogenes. Int J Food Sci Technol, 47, 2460-2467.
  • 38. Yamazaki W, Ishibashi M, Kawahara R, et al (2008): Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus. BMC Microbiol, 8, 163.
  • 39. Yamazaki W, Kumeda Y, Misawa N, et al (2010): Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species. Appl Environ Microbiol, 76, 820-828.
Primary Language en
Subjects Veterinary
Journal Section Research Article
Authors

Orcid: 0000-0002-3712-5021
Author: Yusuf DOĞRUER
Institution: Selçuk University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology
Country: Turkey


Orcid: 0000-0001-8899-4537
Author: A. Ezgi TELLİ (Primary Author)
Institution: Selçuk University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology
Country: Turkey


Dates

Publication Date : September 1, 2020

Bibtex @research article { auvfd603868, journal = {Ankara Üniversitesi Veteriner Fakültesi Dergisi}, issn = {}, eissn = {1308-2817}, address = {}, publisher = {Ankara University}, year = {2020}, volume = {67}, pages = {349 - 355}, doi = {10.33988/auvfd.603868}, title = {Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP}, key = {cite}, author = {Doğruer, Yusuf and Telli̇, A. Ezgi} }
APA Doğruer, Y , Telli̇, A . (2020). Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP . Ankara Üniversitesi Veteriner Fakültesi Dergisi , 67 (4) , 349-355 . DOI: 10.33988/auvfd.603868
MLA Doğruer, Y , Telli̇, A . "Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP" . Ankara Üniversitesi Veteriner Fakültesi Dergisi 67 (2020 ): 349-355 <http://vetjournal.ankara.edu.tr/en/pub/issue/56621/603868>
Chicago Doğruer, Y , Telli̇, A . "Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP". Ankara Üniversitesi Veteriner Fakültesi Dergisi 67 (2020 ): 349-355
RIS TY - JOUR T1 - Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP AU - Yusuf Doğruer , A. Ezgi Telli̇ Y1 - 2020 PY - 2020 N1 - doi: 10.33988/auvfd.603868 DO - 10.33988/auvfd.603868 T2 - Ankara Üniversitesi Veteriner Fakültesi Dergisi JF - Journal JO - JOR SP - 349 EP - 355 VL - 67 IS - 4 SN - -1308-2817 M3 - doi: 10.33988/auvfd.603868 UR - https://doi.org/10.33988/auvfd.603868 Y2 - 2020 ER -
EndNote %0 Ankara Üniversitesi Veteriner Fakültesi Dergisi Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP %A Yusuf Doğruer , A. Ezgi Telli̇ %T Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP %D 2020 %J Ankara Üniversitesi Veteriner Fakültesi Dergisi %P -1308-2817 %V 67 %N 4 %R doi: 10.33988/auvfd.603868 %U 10.33988/auvfd.603868
ISNAD Doğruer, Yusuf , Telli̇, A. Ezgi . "Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP". Ankara Üniversitesi Veteriner Fakültesi Dergisi 67 / 4 (September 2020): 349-355 . https://doi.org/10.33988/auvfd.603868
AMA Doğruer Y , Telli̇ A . Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP. Ankara Univ Vet Fak Derg. 2020; 67(4): 349-355.
Vancouver Doğruer Y , Telli̇ A . Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP. Ankara Üniversitesi Veteriner Fakültesi Dergisi. 2020; 67(4): 349-355.
IEEE Y. Doğruer and A. Telli̇ , "Determination of Vibrio parahaemolyticus in seafoods using direct plate counting, quantitative loop-mediated isothermal amplification and propidium monoazide-qLAMP", Ankara Üniversitesi Veteriner Fakültesi Dergisi, vol. 67, no. 4, pp. 349-355, Sep. 2020, doi:10.33988/auvfd.603868