Biotraceability in Food and Feed Chains--Course Descriptions

Molecular Methods for Salmonella

Overview
Part 1. Methods for identification and characterization of stress response genes
Maj-Britt Nielsen, LIFE, KU, DK, and Gitte Knudsen, IFR, UK

Part 2. Qualitative and quantitative detection methods
Nadine Krämer: Real-Time PCR for qualitative and quantitative detection for Salmonella from slaughterhouse samples
Ralf Dieckmann: Rapid identification of Salmonella isolates by Matrix-Assisted Laser Desorption/Ionisation mass spectrometry

The workshop is focussing on molecular methods for Salmonella useful for understanding bio-traceability processes.

Part 1: The first part is focussing on the study of bacterial stress response. The participants will be introduced to the Gel Cassette model used in BIOTRACER for studying immobilised and planktonic growth of Salmonella. The focus will be on DNA microarrays for transcriptomic analysis including new techniques for transcriptomic analysis and construction of mutants and gfp-promoter fusions using the Lambda Red techniques. In addition, an introduction to phenotypic experiments used to assess the mutants and wildtypes such as in vitro assays in HeLa cells for studying virulence potential of cells and Western blots.

Part 2: A second part is focussing on the qualitative and quantitative methods for the enumeration of Salmonella from food samples. The following topics will be presented: introduction in real-time PCR (qualitative and quantitative), qualitative sensitive detection of Salmonella by real-time PCR, enumeration of salmonellae by real-time PCR, the Most Probable Number method as a cultural reference method.
Another new method for the rapid identification of Salmonella species, subspecies and serovars using Matrix-Assisted Laser Desorption/Ionisation mass spectrometry (MALDI-TOF MS) is presented. This technique has enormous potential for the identification of Salmonella using bacterial colonies in a few minutes.

Pre-workshop Required Reading*
Part 1

Lucchini, S., A. Thompson, and J. C. Hinton (2001). Microarrays for microbiologists. Microbiology 147:1403-1414
Datsenko, K. A. and B. L. Wanner (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A 97:6640-6645
IFR Gel Cassette System (Flyer, IFR).

Part 2

Real-Time PCR Vs. Traditional PCR (Tutorial, Applied Biosystems)
N. A. Saunders (2004) Quantitative Real-Time PCR In: Real-Time PCR: An essential guide (Kirstin Edwards, Julie Logan and Nick Saunders, eds.) Horizon Bioscience, London, chapter 6.
D. Dare (2006) Rapid Bacterial Characterization and Identification by MALDI-TOF Mass Spectrometry. In: Advanced Techniques in Diagnostic Microbiology (Yi-Wei Tang and Charles W. Stratton, eds.) Springer US, 117-133

*Required for PhD students.

Post-workshop Reading Material

Essentials of Real-Time PCR (Applied Biosystems)
Standard Operating Procedure (SOP) for rapid quantification of Salmonella spp. in pig cork borer samples in the BIOTRACER project
Standard Operating Procedure (SOP) for rapid quantification of Salmonella spp. in pig carcass swab samples in the BIOTRACER project
Standard Operating Procedure (SOP) for MPN-PCR method for Salmonella used in the BIOTRACER project
B. Malorny, C. Löfström, M. Wagner, N. Krämer, J. Hoorfar (2008) Enumeration of salmonella bacteria in food and feed samples by real-time PCR for quantitative microbial risk assessment. Appl. Environ. Microbiol., 74:1299-304.
B. Malorny, S. Huehn, R. Dieckmann, N. Krämer, R. Helmuth (2009) Polymerase Chain Reaction for the Rapid Detection and Serovar Identification of Salmonella in Food and Feeding Stuff, Food Anal. Methods, 2: 81-95.
R. Dieckmann, R. Helmuth, M. Erhard, B. Malorny (2008) Rapid classification/identification of salmonellae at the species and subspecies level using whole-cell MALDI-TOF mass spectrometry. Appl. Environ. Microbiol., 74:7767-7778.

Papers
Brocklehurst, T. F., G. A. Mitchell, Y. P. Ridge, R. Seale, and A. C. Smith (1995). The effect of transient temperatures on the growth of Salmonella typhimurium LT2 in gelatin gel. Int. J. Food Microbiol. 27:45-60.
MacLean, D., J. D. Jones, and D. J. Studholme (2009). Application of 'next-generation' sequencing technologies to microbial genetics. Nat. Rev. Microbiol. 7:287-296.
Hautefort, I., M. J. Proenca, and J. C. Hinton (2003). Single-copy green fluorescent protein gene fusions allow accurate measurement of Salmonella gene expression in vitro and during infection of mammalian cells. Appl. Environ. Microbiol. 69:7480-7491
Wang, Q., Y. Zhao, M. McClelland, and R. M. Harshey (2007). The RcsCDB signaling system and swarming motility in Salmonella enterica serovar Typhimurium: dual regulation of flagellar and SPI-2 virulence genes. J. Bacteriol. 189:8447-8457.

Protocols
- Growth experiment in the Gel Cassette system
- Harvest cells for RNA from the Gel Cassette system
- RNA extraction and purification
- Direct labelling of DNA
- Direct labelling of RNA
- Labelling protocol for reduced amounts of RNA
- Lambda-red knockout protocol
- Microarray glass slide blocking
- Microarray Hybridisations
- P22 Transduction
- Determination of bacterial invasion and adhesion of HeLa cell line