Multiplex T-RFLP Allows for Increased Target Number and Specificity: Detection of Salmonella enterica and Six Species of Listeria in a Single Test

Abstract

A multiplex T-RFLP test was developed to detect and identify Salmonella enterica and all six species of Listeria inoculated into milk at minimal levels. Extensive in silico analysis was used to design a fifteen-primer, six-amplimer methodology and in vitro application showed target organism DNA, when amplified individually, yielded the predicted terminal restriction fragments (TRFs) following digestion. Non-target organisms were either not-amplified or yielded TRFs which did not interfere with target identification. Multiple target DNA analysis gave over 86% detection of total TRFs predicted, and this was improved to over 90% detection of total TRFs predicted when only two target DNA extracts were combined analysed. Co-inoculation of milk with five strains each of the target species of S. enterica and L. monocytogenes, along with five strains of the non-target species E. coli was followed by enrichment in SEL medium for M-TRFLP analysis. This allowed for detection of both target species in all samples, with detection of one S. enterica and two Listeria TRFs in all cases, and detection of a second S. enterica TRF in 91% of cases. This was from an initial inoculum of <5 cfu per 25 ml milk with a background of competing E. coli present, and gave a result from sampling of under 20 hours. The ability to increase target species number without loss of sensitivity means that extensive screening can be performed at reduced cost due to a reduction in the number of tests required.

Published in

PLoS ONE
2012, volume: 7, number: 8, article number: e43672
Publisher: PUBLIC LIBRARY SCIENCE 1160 BATTERY STREET, STE 100, SAN FRANCISCO, CA 94111 USA

SLU Authors

• Campbell, Colin

  • Department of Soil and Environment, Swedish University of Agricultural Sciences
    
  • The James Hutton Institute

UKÄ Subject classification

Microbiology

Publication identifier

• DOI: https://doi.org/10.1371/journal.pone.0043672

Permanent link to this page (URI)

https://res.slu.se/id/publ/38799