title: 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.
authors.affiliation: James Hutton Institute, Craigiebuckler, Aberdeen, United Kingdom. geoff.elliott@hutton.ac.uk
authors.forename: Geoffrey N
authors.initials: GN
authors.lastname: Elliott
authors.forename: Nadine
authors.initials: N
authors.lastname: Thomas
authors.forename: Marion
authors.initials: M
authors.lastname: Macrae
authors.forename: Colin D
authors.initials: CD
authors.lastname: Campbell
authors.forename: Iain D
authors.initials: ID
authors.lastname: Ogden
authors.forename: Brajesh K
authors.initials: BK
authors.lastname: Singh
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D000818:Animals; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D008087:Listeria / Q000235:genetics / Q000302:isolation & purification*; D008892:Milk / Q000737:chemistry; D012150:Polymorphism, Restriction Fragment Length / Q000235:genetics*; D019779:Salmonella enterica / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial