Three pre-PCR processing strategies for the detection and/or quantification of Salmonella in naturally contaminated soy bean meal were evaluated

Andersson

Aprodu

Binter

Cocolin

D'Aoust

Davies

Fukushima

Gorski

Halvorson

Hedman

International Organisation for Standardization

Jacobson

Jarvis

Josefsen

Koyuncu

Krämer

Le Hello

Lebaron

Lindqvist

Löfström

Maciorowski

Malorny

Nordic Committee on Food Analysis

Opel

Pavic

Pertoft

Rossmanith

Rostagno

Sauli

Singer

Smeltzer

Soria

Wesche

Wierup

Wolffs

Schelin, Jenny

Andersson, Gunnar

Vigre, Håkan

Norling, Börje

Häggblom, Per

Hoorfar, Jeffrey

Rådström, Peter

Löfström, Charlotta

Lund University Publications

Evaluation of pre-PCR processing approaches for enumeration of Salmonella enterica in naturally contaminated animal feed.

Schelin, J.

Andersson, G.

Norling, B.

Häggblom, P.

Rådström, P.

Online Research Database In Technology

                       General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  • 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.     Downloaded from orbit.dtu.dk on: Dec 20, 2017Evaluation of pre-PCR processing approaches for enumeration of Salmonella entericain naturally contaminated animal feedLöfström, Charlotta; Schelin, J.; Andersson, G.; Vigre, Håkan; Norling, B.; Häggblom, P.; Hoorfar,Jeffrey; Rådström, P.Publication date:2013Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Löfström, C., Schelin, J., Andersson, G., Vigre, H., Norling, B., Häggblom, P., ... Rådström, P. (2013). Evaluationof pre-PCR processing approaches for enumeration of Salmonella enterica in naturally contaminated animalfeed. Poster session presented at MedVetNet Association International Scientific Conference 2013, Lyngby,Denmark.Evaluation of pre-PCR processing approaches for enumeration of Salmonella enterica in naturally contaminated animal feed Aim To evaluate 3 pre-PCR processing strategies: 1) flotation-qPCR (modified from [1]) 2) MPN-PCR (modified from [2]) 3) qualitative culture enrichment PCR [2] for the detection and/or quantification of Salmonella in naturally contaminated soy bean meal.  Introduction Animal feed might serve as a reservoir of Salmonella contributing to the spread into the food chain. Levels of Salmonella in feed samples are low, bacteria are unevenly distributed and stressed and could therefore be hard to recover using standard culture-based methods. Due to this, there is a need for accurate, sensitive, rapid and user-friendly sample preparation methods prior to molecular analyses. Moreover, to facilitate quantitative risk assessment in the feed production chain, there is a need to enumerate Salmonella in naturally contaminated feed.   Methods C. Löfströma, J. Schelinb, G. Anderssonc, H. Vigrea, B. Norlingd,  P. Häggblomc, J. Hoorfara, & P. Rådströmb aNational Food Institute, Technical University of Denmark, Søborg; bApplied Microbiology, Lund University, Sweden;  cNational Veterinary Institute (SVA), Uppsala, Sweden; d Quintessence Research AB, Alunda, Sweden  Conclusions • The 3 methods provide possibilities to assess the prevalence of Salmonella in feed, as well as the numbers of culturable, and non-culturable cells • Differences in results could be due to non-culturable Salmonella and/or a heterogeneous distribution of Salmonella in the feed  Results Out of the 15 bags analyzed 6, 15 and 9 were positive for Salmonella with flotation-qPCR, MPN-PCR and culture enrichment PCR, respectively (Table 1). Enumeration resulted in values of 1.8×102-7.8×103 CFU/g (flotation-qPCR) and 0.024 to >5.2 MPN/g (MPN-PCR) (Figure 2).      References 1. Löfström et al. (2011)  IJFM 145 Suppl 1:S103-109  2. Löfström et al  (2012) Vet. Microbiol. 158(3-4):431-435. 3. Malorny et al (2004) AEM, 70(12):7046-52.  Acknowledgements  This study was financially supported by the EU project BIOTRACER (contract no. 036272) and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS).  Table 1. Comparison of qualitative results with the three methods for the analysis of naturally contaminated soya bean meal samples. Method Qualitative result (+/-) for indicated bag with soya bean meal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Flotation-qPCR + + - + - - + - - - - + - + - MPN-PCR + + + + + + + + + + + + + + + Culture enrichment PCR + + + + - + - - + - + + - + - Figure 2. Comparison of enumeration results with flotation-qPCR and MPN-PCR for the analysis of naturally contaminated soya bean meal samples. Real-time PCR ttrRSBCA locus [2-3] Salmonella positive soya bean meal (15 bags à 250 g, each representing a 2,000 tons sublot from a 30,000 tons lot)) Incubation at 37 ºC, 16 ± 2 h Magnetic beads based  DNA extraction 200 ml 50 ml 10 ml 2 ml 2.5 g: Flotation- qPCR [1] 50 g: MPN- PCR 25 g: Culture enrichment  PCR [2] Homogenization in  BPW 1:10 • Addition of two layers of density media • Centrifugation Photo: By Roosewelt Pinheiro/Abr [CC-BY-3.0-br], via Wikimedia Commons Figure 1. Overview of the three pre-PCR processing strategies that were investigated. Bags of naturally contaminated soya bean meal (n = 15) were analyzed in parallel with the three methods. 

Evaluation of pre-PCR processing approaches for enumeration of Salmonella enterica in naturally contaminated animal feed

Schelin, Jenny,

Andersson, Gunnar,

Vigre, Håkan,

Norling, Börje,

Häggblom, Per,

Hoorfar, Jeffrey,

Löfström, Charlotta,

Swepub

Anderson, Gunnar

Three pre‐PCR processing strategies for the detection and/or quantification of Salmonella in naturally contaminated soya bean meal were evaluated. Methods included: (i) flotation‐qPCR [enumeration of intact Salmonella cells prior to quantitative PCR (qPCR)], (ii) MPN‐PCR (modified most probable number method combined with qPCR) and (iii) qualitative culture enrichment PCR. The limit of quantification was 1·8 × 102 CFU g−1 (flotation‐qPCR) and 0·02 MPN g−1 (MPN‐PCR). Fifteen naturally contaminated Salmonella positive soya bean meal samples from one lot were analysed in parallel with the three methods, using 2·5, 50 and 25 g of feed, respectively, resulting in detection of Salmonella in 6, 15 and 9 bags. Enumeration resulted in 1·8 × 102–7·8 × 103 CFU g−1 (flotation‐qPCR) and 0·024 to &gt;5·2 MPN g−1 (MPN‐PCR). Except for differences in methodology, results obtained with the three techniques could be due to the presence of nonculturable Salmonella and/or a heterogeneous distribution of Salmonella in the material. The evaluated methods provide different possibilities to assess the prevalence of Salmonella in feed, together with the numbers of culturable, as well as nonculturable cells, and can be applied to generate data to allow more accurate quantitative microbial risk assessment for Salmonella in the feed chain

J. Schelin

G. Andersson

H. Vigre

B. Norling

P. Häggblom

J. Hoorfar

P. Rådström

C. Löfström

Crossref

Journal of Applied Microbiology

Evaluation of pre-PCR processing approaches for enumeration of<i>Salmonella enterica</i>in naturally contaminated animal feed

http://orbit.dtu.dk/ws/files/55645542/130624%20poster%20L%C3%B6fstr%C3%B6m%20et%20al%20MedVetNet2013%20final.pdf

Evaluation of pre-PCR processing approaches for enumeration of Salmonella enterica in naturally contaminated animal feed

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