Impact of various killing methods on EMA/PMA-qPCR efficacy

Vondrakova, Lucie; Turonova, Hana; Scholtz, Vladimír; Pazlarová, J.; Demnerova, Kateřina

doi:10.1016/j.foodcont.2017.09.013

Cited by 21 publications

(8 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Different inactivation methods were recently shown to affect the efficiency and accuracy Running Header: Long-amplicon PMA-qPCR assay for enumeration of viable L. monocytogenes 34 of short-amplicon PMA-qPCR and EMA-qPCR to detect viable Campylobacter (Vondrakova et al, 2018).…”

Section: Long-amplicon Pma-qpcr As a Methods To Discriminate Between Vmentioning

confidence: 99%

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

2020

View full text Add to dashboard Cite

 Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

show abstract

Section: Long-amplicon Pma-qpcr As a Methods To Discriminate Between Vmentioning

confidence: 99%

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

2020

View full text Add to dashboard Cite

show abstract

“…The theoretical mode of action includes the selective entrance of PMA into dead bacterial cells via their compromised-membranes and the DNA cleavage upon photoactivation that prohibits its subsequent amplification during qPCR ( Nocker et al, 2006 ; Wagner et al, 2008 ; Varma et al, 2009 ). However, applications of v-qPCR protocols with PMA (PMA-qPCR) on meat samples have encountered various challenges and limitations attributed to a rather conditional and not absolute suppression of qPCR signals originating from dead Campylobacter bacteria (false-positive signals) due to a complex set of parameters including experimental, target and sample features ( Nocker et al, 2006 ; Pacholewicz et al, 2013 ; Krüger et al, 2014 ; Duarte et al, 2015 ; Vondrakova et al, 2018 ). The development of a PMA-qPCR assay utilizing spheroplast formation as a pretreatment for enhancing the selective entrance of PMA to dead cells and an internal sample process control (ISPC), unique for any given sample, that could address false-positive signals originating from dead cells, has only recently been achieved using C. coli as a bacterial model ( Lazou et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Method-Dependent Implications in Foodborne Pathogen Quantification: The Case of Campylobacter coli Survival on Meat as Comparatively Assessed by Colony Count and Viability PCR

et al. 2021

View full text Add to dashboard Cite

The aim of the present study was to address method-dependent implications during the quantification of viable Campylobacter coli cells on meat over time. Traditional colony counting on selective and non-selective culture media along with an optimized viability real-time PCR utilizing propidium monoazide-quantitative PCR (PMA-qPCR), spheroplast formation and an internal sample process control (ISPC), were comparatively evaluated for monitoring the survival of C. coli on fresh lamb meat during refrigeration storage under normal atmospheric conditions. On day zero of three independent experiments, lamb meat pieces were artificially inoculated with C. coli and then stored under refrigeration for up to 8 days. Three meat samples were tested on different days and the mean counts were determined per quantification method. An overall reduction of the viable C. coli on lamb meat was observed regardless of the applied quantification scheme, but the rate of reduction followed a method-dependent pattern, the highest being observed for colony counting on modified charcoal cefoperazone deoxycholate agar (mCCDA). Univariate ANOVA indicated that the mean counts of viable C. coli using PMA-qPCR were significantly higher compared to Columbia blood agar (CBA) plating (0.32 log10 cell equivalents, p = 0.015) and significantly lower when mCCDA was compared to CBA plating (0.88 log10 CFU, p < 0.001), indicating that selective culture on mCCDA largely underestimated the number of culturable cells during the course of meat storage. PMA-qPCR outperformed the classical colony counting in terms of quantifying both the culturable and viable but non-culturable (VBNC) C. coli cells, which were generated over time on meat and are potentially infectious and equally important from a public health perspective as their culturable counterparts.

show abstract

“…For this purpose, bacterial cells were lysed by heat for 5 min and directly applied in the amplification reaction. Direct PCR is increasingly utilized for clinical, forensic, agricultural and genetic applications since it saves time and considerably reduces costs by lowering the number of necessary chemicals [35].…”

Section: Discussionmentioning

confidence: 99%

A Model System for Sensitive Detection of Viable E. coli Bacteria Combining Direct Viability PCR and a Novel Microarray-Based Detection Approach

et al. 2021

View full text Add to dashboard Cite

We established an innovative approach that included direct, viability, and nested PCR for rapid and reliable identification of the fecal indicator organism Escherichia coli (E. coli). Direct PCR enabled successful amplification of the target uidA gene, omitting a prior DNA isolation or purification step. Furthermore, we applied viability PCR (v-PCR) to ensure the detection of only relevant viable bacterial cells. The principle involves the binding of propidium monoazide (PMA), a selective nucleic acid intercalating dye, to accessible DNA of heat killed bacteria cells and, consequently, allows viable and heat killed E. coli cells to be discriminated. To ensure high sensitivity, direct v-PCR was followed by a nested PCR step. The resulting amplicons were analyzed by a rapid 30 min microarray-based DNA hybridization assay for species-specific DNA detection of E. coli. A positive signal was indicated by enzymatically generated silver nanoparticle deposits, which served as robust endpoint signals allowing an immediate visual readout. The presented novel protocol allows the detection of 1 × 101 viable E. coli cells per PCR run.

show abstract

Impact of various killing methods on EMA/PMA-qPCR efficacy

Cited by 21 publications

References 23 publications

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

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

Method-Dependent Implications in Foodborne Pathogen Quantification: The Case of Campylobacter coli Survival on Meat as Comparatively Assessed by Colony Count and Viability PCR

A Model System for Sensitive Detection of Viable E. coli Bacteria Combining Direct Viability PCR and a Novel Microarray-Based Detection Approach

Contact Info

Product

Resources

About