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

• Published in: PloS one Vol. 7; no. 8; p. e43672
• Main Authors: Elliott, Geoffrey N., Thomas, Nadine, MacRae, Marion, Campbell, Colin D., Ogden, Iain D., Singh, Brajesh K.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 24.08.2012; Public Library of Science (PLoS)
• Subjects: Amplified Fragment Length Polymorphism Analysis - methods; Analysis; Animals; Bacteria; Biology; Colony Count, Microbial; Deoxyribonucleic acid; DNA; DNA, Bacterial - analysis; DNA, Bacterial - genetics; E coli; Enterobacter; Escherichia coli; Food; Genes; Genetic aspects; Identification; Identification and classification; In vitro methods and tests; Inoculation; Inoculum; Listeria; Listeria - genetics; Listeria - isolation & purification; Listeria monocytogenes; Medicine; Microbiology; Mikrobiologi; Milk; Milk - chemistry; Multiplexing; Pathogens; Polymorphism, Restriction Fragment Length - genetics; Restriction fragment length polymorphism analysis; Salmonella; Salmonella enterica; Salmonella enterica - genetics; Salmonella enterica - isolation & purification; Sensitivity and Specificity; Shigella; Species; Strains (organisms); Studies; Target detection; Target recognition; Vegetation; Veterinary Science; Yersinia; Sweden; United Kingdom > UK
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0043672

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.