Multiplex T-RFLP allows for increased target number and specificity: detection of Salmonella enterica and six species of Listeria in a single test.

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, whe...

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Autores principales:	Geoffrey N Elliott, Nadine Thomas, Marion Macrae, Colin D Campbell, Iain D Ogden, Brajesh K Singh
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b6be21108d864882abe6500d60a72db7
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spelling	oai:doaj.org-article:b6be21108d864882abe6500d60a72db72021-11-18T07:07:43ZMultiplex T-RFLP allows for increased target number and specificity: detection of Salmonella enterica and six species of Listeria in a single test.1932-620310.1371/journal.pone.0043672https://doaj.org/article/b6be21108d864882abe6500d60a72db72012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22937073/?tool=EBIhttps://doaj.org/toc/1932-6203A 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.Geoffrey N ElliottNadine ThomasMarion MacraeColin D CampbellIain D OgdenBrajesh K SinghPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 8, p e43672 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Geoffrey N Elliott Nadine Thomas Marion Macrae Colin D Campbell Iain D Ogden Brajesh K Singh Multiplex T-RFLP allows for increased target number and specificity: detection of Salmonella enterica and six species of Listeria in a single test.
description	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.
format	article
author	Geoffrey N Elliott Nadine Thomas Marion Macrae Colin D Campbell Iain D Ogden Brajesh K Singh
author_facet	Geoffrey N Elliott Nadine Thomas Marion Macrae Colin D Campbell Iain D Ogden Brajesh K Singh
author_sort	Geoffrey N Elliott
title	Multiplex T-RFLP allows for increased target number and specificity: detection of Salmonella enterica and six species of Listeria in a single test.
title_short	Multiplex T-RFLP allows for increased target number and specificity: detection of Salmonella enterica and six species of Listeria in a single test.
title_full	Multiplex T-RFLP allows for increased target number and specificity: detection of Salmonella enterica and six species of Listeria in a single test.
title_fullStr	Multiplex T-RFLP allows for increased target number and specificity: detection of Salmonella enterica and six species of Listeria in a single test.
title_full_unstemmed	Multiplex T-RFLP allows for increased target number and specificity: detection of Salmonella enterica and six species of Listeria in a single test.
title_sort	multiplex t-rflp allows for increased target number and specificity: detection of salmonella enterica and six species of listeria in a single test.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/b6be21108d864882abe6500d60a72db7
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Multiplex T-RFLP allows for increased target number and specificity: detection of Salmonella enterica and six species of Listeria in a single test.

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