A long-amplicon quantitative PCR assay with propidium monazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments

Martin Laage Kragh, Mikala Thykier, Lisbeth Truelstrup Hansen*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33% RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92%, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102%, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.
Original languageEnglish
Article number103310
JournalFood Microbiology
Volume86
Number of pages10
ISSN0740-0020
DOIs
Publication statusPublished - 2020

Keywords

  • Amplicon length
  • Viability
  • Biofilm
  • Stainless steel
  • PMA-qPCR

Cite this

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title = "A long-amplicon quantitative PCR assay with propidium monazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments",
abstract = "The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33{\%} RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92{\%}, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102{\%}, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.",
keywords = "Amplicon length, Viability, Biofilm, Stainless steel, PMA-qPCR",
author = "Kragh, {Martin Laage} and Mikala Thykier and Hansen, {Lisbeth Truelstrup}",
year = "2020",
doi = "10.1016/j.fm.2019.103310",
language = "English",
volume = "86",
journal = "Food Microbiology",
issn = "0740-0020",
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}

A long-amplicon quantitative PCR assay with propidium monazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments. / Kragh, Martin Laage; Thykier, Mikala; Hansen, Lisbeth Truelstrup.

In: Food Microbiology, Vol. 86, 103310, 2020.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - A long-amplicon quantitative PCR assay with propidium monazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments

AU - Kragh, Martin Laage

AU - Thykier, Mikala

AU - Hansen, Lisbeth Truelstrup

PY - 2020

Y1 - 2020

N2 - The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33% RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92%, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102%, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.

AB - The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33% RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92%, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102%, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.

KW - Amplicon length

KW - Viability

KW - Biofilm

KW - Stainless steel

KW - PMA-qPCR

U2 - 10.1016/j.fm.2019.103310

DO - 10.1016/j.fm.2019.103310

M3 - Journal article

VL - 86

JO - Food Microbiology

JF - Food Microbiology

SN - 0740-0020

M1 - 103310

ER -