219
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      New Approaches on Quantification of Campylobacter jejuni in Poultry Samples: The Use of Digital PCR and Real-time PCR against the ISO Standard Plate Count Method

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C. jejuni suspension with a known number of bacterial cells. The observed CFU/g values by qPCR correlated greatly with the expected values and qPCR showed good performance with the reliable limit of detection (rLOD) and limit of quantification (LOQ) of three and 31 target copies per reaction, respectively. However, both rLOD (1219 CFU/g) and LOQ (12,523 CFU/g) were beyond the EFSA-proposed critical limit of 500–1,000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1,000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1,000 CFU/g in 24/75) samples by qPCR. A strong and statistically significant correlation was observed between qPCR and dPCR. Both PCR-based methods correlated significantly with the plate count method; however, the correlation was moderate. Using the Bland–Altman analysis, an average agreement was noted between all three methods, although with a large standard deviation. A significant bias toward overestimation in dPCR was observed, probably due to the relatively high number of false positive calls. The linear dynamic range was comparable in both PCR-based methods; however, qPCR proved to be more suitable for routine use. In the future, the establishment of a reliable molecular quantification of C. jejuni in poultry samples showing a wide range of contamination levels down to the proposed critical limit is needed to enable time- and cost-effective surveillance throughout all stages in the food production chain. As both rLOD and LOQ were beyond this limit, a modification of the procedure is suggested to include less sample dilution prior to DNA extraction to enable PCR-based quantification of C. jejuni at the proposed microbiological criteria.

          Related collections

          Most cited references40

          • Record: found
          • Abstract: found
          • Article: not found

          Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification.

          The ideal scenario in most applications of microbial diagnostics is that only viable cells are detected. Bacteria were traditionally considered viable when they could be cultured, whereas today's viability concept tends to be alternatively based on the presence of some form of metabolic activity, a positive energy status, responsiveness, detection of RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane. The latter criterion, although conservative, was the focus of one of the most successful recent approaches to detect viable cells in combination with DNA amplification techniques. The technology is based on sample treatment with the photoactivatable, and cell membrane impermeant, nucleic acid intercalating dyes ethidium monoazide (EMA) or propidium monoazide (PMA) followed by light exposure prior to extraction of DNA and amplification. Light activation of DNA-bound dye molecules results in irreversible DNA modification and subsequent inhibition of its amplification. Sample pretreatment with viability dyes has so far been mainly used in combination with PCR (leading to the term viability PCR, v-PCR), and increasingly with isothermal amplification method. The principle is not limited to bacteria, but has also successfully been applied to fungi, protozoa and viruses. Despite the success of the method, some practical limitations have been identified, especially when applied to environmental samples. In part they can be minimized by choice of experimental parameters and conditions adequate for a particular sample. This review summarizes current knowledge and presents aspects which are important when designing experiments employing viability dyes. Copyright © 2012 Elsevier B.V. All rights reserved.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Dead or alive: molecular assessment of microbial viability.

            Nucleic acid-based analytical methods, ranging from species-targeted PCRs to metagenomics, have greatly expanded our understanding of microbiological diversity in natural samples. However, these methods provide only limited information on the activities and physiological states of microorganisms in samples. Even the most fundamental physiological state, viability, cannot be assessed cross-sectionally by standard DNA-targeted methods such as PCR. New PCR-based strategies, collectively called molecular viability analyses, have been developed that differentiate nucleic acids associated with viable cells from those associated with inactivated cells. In order to maximize the utility of these methods and to correctly interpret results, it is necessary to consider the physiological diversity of life and death in the microbial world. This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
              Bookmark
              • Record: found
              • Abstract: not found
              • Article: not found

              The European Union summary report on trends and sources of zoonoses, zoonotic agents and food‐borne outbreaks in 2014

              (2015)
                Bookmark

                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                02 March 2017
                2017
                : 8
                : 331
                Affiliations
                [1] 1Veterinary Faculty, Institute of Microbiology and Parasitology, University of Ljubljana Ljubljana, Slovenia
                [2] 2Veterinary Faculty, Institute of Food Safety, Feed and Environment, University of Ljubljana Ljubljana, Slovenia
                Author notes

                Edited by: Petr Kralik, Veterinary Research Institute, India

                Reviewed by: Braulio Esteve-Zarzoso, Universidad Rovira i Virgili, Spain; Mehrdad Mark Tajkarimi, University of North Carolina at Greensboro, USA

                *Correspondence: Darja Kušar darja.kusar@ 123456vf.uni-lj.si

                This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2017.00331
                5332403
                28303130
                ce0ea781-ac91-4d07-8c05-b0e8be43cbc4
                Copyright © 2017 Papić, Pate, Henigman, Zajc, Gruntar, Biasizzo, Ocepek and Kušar.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 07 October 2016
                : 17 February 2017
                Page count
                Figures: 4, Tables: 4, Equations: 0, References: 50, Pages: 13, Words: 9115
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                campylobacter jejuni,poultry,quantification,plate counting,qpcr,dpcr
                Microbiology & Virology
                campylobacter jejuni, poultry, quantification, plate counting, qpcr, dpcr

                Comments

                Comment on this article