Molecular analysis

The National Salmonella, Shigella & Listeria Reference Laboratory provides a molecular typing service on isolates of interest of Salmonella enterica, Shigella species, and Listeria monocytogenes.

Pulse Field Gel Electrophoresis (PFGE)

Summary

An isolate(s) is cultured on to an agar plate, and a liquid suspension is made of colonies that grow on the plate.  Measured amounts of the bacterial suspension are mixed with small amounts of agarose.  These agarose 'plugs' are treated in such a way to break down the wall of the bacterial cells embedded within.  When the wall of the cells are broken down the cells are washed and then mixed with an enzyme (endonuclease) which digests the cell DNA into many fragments.  Gel electrophoresis is then set-up and digested cell 'plugs' are embedded to the top of the gel.  An electrical pulsed field is created and applied to gel in a buffered environment.   After 18hr distinct DNA banding patterns may be visualised with staining and UV light. 

 
Figure: PFGE of isolates of Salmonella enterica serotype Typhimurium

We comply with PulseNet typing protocol which uses a standardised Pulse field electrophoresis (PFGE) method to subtype and analyse certain groups of bacterial isolates.

NSSLRL is connected to the European Pulse-Net network where molecular patterns are sent to a centralised server database for comparison with other isolates.

 
Figure: PFGE of isolates of Listeria spp.

BIONUMERICS ANALYSIS APPLICATION

BioNumerics is software from Applied Maths BVBA (Sint-Marteens-Latem, Belgium) to analyse and compare *.tif images and *.bdl files from PFGE gels.

The following picture is representative of dendogram analysis that is produced from this software.

Multi locus variable number of tandem repeat analysis (MLVA)

Multi locus variable number of tandem repeat analysis (MLVA) is based on repetitive DNA sequences called tandem repeats which mutate at a high rate. The NSRL performs MLVA on S.Typhimurium isolates while an MLVA scheme for S.Enteritidis is currently being evaluated. For S.Typhimurium repetitive elements of 5 genes are analysed. A multiplex Polymerase Chain Reaction (PCR) is performed using flourescent primers to amplify the regions of interest. The PCR products are then analysed by a sequencer to produce accurate size measurements. The peak sizes allow the number of tandem regions in each region be estinated. The results of the 5 genes are combined to give an MLVA pattern or allele string for each isolate, e.g. 03-14-06-12-311. MLVA patterns from different isolates can be compared. MLVA allows discrimination between isolates which would otherwise appear homogenous, e.g. S.Typhimurium isolates of the DT104 family which appear clonal using PFGE. This technique helps in the detection of Salmonella outbreaks and also in tracing links between animal and food sources and human infections.

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