Quantification of Viable in Cold-Smoked Salmon Using PMA/PMAxx-qPCR.

Agnès Bouju-Albert, Sabrina Saltaji, Xavier Dousset, Hervé Prévost, Emmanuel Jaffrès
Author Information
  1. Agnès Bouju-Albert: UMR 1014, Secalim, INRAE, Oniris, Nantes, France.
  2. Sabrina Saltaji: UMR 1014, Secalim, INRAE, Oniris, Nantes, France.
  3. Xavier Dousset: UMR 1014, Secalim, INRAE, Oniris, Nantes, France.
  4. Hervé Prévost: UMR 1014, Secalim, INRAE, Oniris, Nantes, France.
  5. Emmanuel Jaffrès: UMR 1014, Secalim, INRAE, Oniris, Nantes, France.

Abstract

The aim of this study was to develop a rapid and accurate PMA-qPCR method to quantify viable in cold-smoked salmon. is one of the main food spoilage bacteria. Among seafood products, cold-smoked salmon is particularly impacted by spoilage. Specific and sensitive tools that detect and quantify this bacterium in food products are very useful. The culture method commonly used to quantify is time-consuming and can underestimate cells in a viable but not immediately culturable state. We designed a new PCR primer set from the single-copy gene. QPCR efficiency and specificity were compared with two other published primer sets targeting the and genes. The viability dyes PMA or PMAxx were combined with qPCR and compared with these primer sets on viable and dead cells in BHI broth and smoked salmon tissue homogenate (SSTH). The three primer sets displayed similar specificity and efficiency. The efficiency of new designed qPCR on viable cells in SSTH was 103.50%, with a linear determination coefficient (r) of 0.998 and a limit of detection of 4.04 log CFU/g. Using the three primer sets on viable cells, no significant difference was observed between cells treated or untreated with PMA or PMAxx. When dead cells were used, both viability dyes suppressed DNA amplification. Nevertheless, our results did not highlight any difference between PMAxx and PMA in their efficiency to discriminate viable from unviable cells in cold-smoked salmon. Thus, this study presents a rapid, specific and efficient -PMA-qPCR method validated in cold-smoked salmon to quantify viable in foods.

Keywords

References

  1. Appl Environ Microbiol. 2016 Jun 13;82(13):3928-3939 [PMID: 27107120]
  2. Front Microbiol. 2020 May 04;11:673 [PMID: 32431672]
  3. Stand Genomic Sci. 2018 Oct 10;13:22 [PMID: 30338025]
  4. Appl Environ Microbiol. 2010 Aug;76(15):5097-104 [PMID: 20562292]
  5. Food Microbiol. 2012 May;30(1):316-20 [PMID: 22265318]
  6. Appl Environ Microbiol. 2013 Apr;79(8):2612-9 [PMID: 23396343]
  7. Int J Food Microbiol. 2009 Apr 30;131(1):20-9 [PMID: 18573557]
  8. Poult Sci. 2018 May 1;97(5):1706-1711 [PMID: 29471351]
  9. FEMS Microbiol Ecol. 2009 Jun;68(3):351-62 [PMID: 19302550]
  10. Anal Bioanal Chem. 2020 Apr;412(9):2009-2023 [PMID: 32052066]
  11. J Dairy Sci. 2017 Nov;100(11):8804-8813 [PMID: 28865862]
  12. Appl Environ Microbiol. 2007 Jan;73(1):278-88 [PMID: 17071787]
  13. Food Microbiol. 2012 May;30(1):173-9 [PMID: 22265298]
  14. J Dairy Sci. 2018 Jun;101(6):4936-4943 [PMID: 29605335]
  15. Int J Food Microbiol. 2011 Jun 30;147(3):195-202 [PMID: 21531471]
  16. Food Microbiol. 2015 Feb;45(Pt A):45-53 [PMID: 25481061]
  17. Appl Environ Microbiol. 2007 Dec;73(24):8028-31 [PMID: 17933922]
  18. Appl Environ Microbiol. 1996 Mar;62(3):1102-6 [PMID: 8975603]
  19. Meat Sci. 2008 Jan;78(1-2):77-89 [PMID: 22062098]
  20. Front Microbiol. 2017 Feb 02;8:108 [PMID: 28210243]
  21. Microbiology (Reading). 2019 Jun;165(6):593-610 [PMID: 30843781]
  22. Microorganisms. 2019 May 13;7(5): [PMID: 31086084]
  23. J Food Sci. 2011 May;76(4):M234-7 [PMID: 22417362]
  24. J Food Prot. 2020 Nov 1;83(11):1852-1862 [PMID: 32556209]
  25. J Microbiol Methods. 2012 Sep;90(3):262-6 [PMID: 22677606]
  26. Int J Food Microbiol. 2016 Jul 16;229:1-6 [PMID: 27085970]
  27. Front Microbiol. 2016 Nov 22;7:1833 [PMID: 27920757]
  28. J Appl Microbiol. 2014 Jan;116(1):1-13 [PMID: 24119073]
  29. Food Microbiol. 2019 Aug;81:22-31 [PMID: 30910085]
  30. Lett Appl Microbiol. 2014 Sep;59(3):263-71 [PMID: 24820436]
  31. BMC Bioinformatics. 2012 Jun 18;13:134 [PMID: 22708584]
  32. Front Microbiol. 2018 Aug 02;9:1747 [PMID: 30116230]
  33. Int J Food Microbiol. 2009 Sep 15;134(3):230-6 [PMID: 19651454]
  34. Front Microbiol. 2020 Sep 22;11:581201 [PMID: 33072053]
  35. Soc Appl Bacteriol Symp Ser. 1992;21:103S-14S [PMID: 1502596]
  36. BMC Microbiol. 2015 Aug 16;15:164 [PMID: 26276157]
  37. Int J Food Microbiol. 2006 Oct 15;112(1):51-61 [PMID: 16949172]
  38. J Dairy Sci. 2015 Mar;98(3):1625-33 [PMID: 25582587]
  39. Genome Announc. 2017 Nov 22;5(47): [PMID: 29167264]
  40. Int J Food Microbiol. 2001 Jun 15;66(3):175-84 [PMID: 11428576]
  41. Front Microbiol. 2020 Jun 17;11:1107 [PMID: 32625171]
  42. PLoS One. 2019 Aug 23;14(8):e0221119 [PMID: 31442247]
  43. Food Microbiol. 2011 Aug;28(5):848-61 [PMID: 21569926]
  44. Lett Appl Microbiol. 2018 Jul;67(1):79-88 [PMID: 29665023]
  45. ISME J. 2015 May;9(5):1105-18 [PMID: 25333463]
  46. J Appl Microbiol. 2012 Nov;113(5):1014-26 [PMID: 22747964]
  47. PLoS One. 2018 May 3;13(5):e0196525 [PMID: 29723290]
  48. J Agric Food Chem. 2006 May 17;54(10):3604-11 [PMID: 19127732]
  49. J Appl Microbiol. 2005;99(1):66-76 [PMID: 15960666]

Word Cloud

Created with Highcharts 10.0.0viablecellssalmonprimerquantifycold-smokedefficiencysetsPMAmethodspoilagePMAxxqPCRstudyrapidfoodproductsuseddesignednewgenespecificitycomparedviabilitydyesdeadsmokedSSTHthreeUsingdifferenceaimdevelopaccuratePMA-qPCRonemainbacteriaAmongseafoodparticularlyimpactedSpecificsensitivetoolsdetectbacteriumusefulculturecommonlytime-consumingcanunderestimateimmediatelyculturablestatePCRsetsingle-copyQPCRtwopublishedtargetinggenescombinedBHIbrothtissuehomogenatedisplayedsimilar10350%lineardeterminationcoefficientr0998limitdetection404logCFU/gsignificantobservedtreateduntreatedsuppressedDNAamplificationNeverthelessresultshighlightdiscriminateunviableThuspresentsspecificefficient-PMA-qPCRvalidatedfoodsQuantificationViableCold-SmokedSalmonPMA/PMAxx-qPCRBrochothrixthermosphactaPMAxx-basedrpoC

Similar Articles

Cited By