Terminal Restriction Fragment (TRF) detection for DNA extraction and post-amplification clean-up protocols for inoculated milk experiments.

<p>A: TRFs detected for both <i>S. enterica</i> (SE) and <i>L. monocytogenes</i> (LM), and are indicated as described in the legend, with three SE TRFs and five LM TRFs expected for all samples. TRFs detected are shown for the mean averages of three individual replicates in each case. Error bars shown are +/-1SD for both TRFs detected values. Milk was inoculated with five strains each of <i>S. enterica</i> (SE), <i>E. coli</i> (EC) and <i>L. monocytogenes</i> (LM) at 4.4, 3.2 and 5.4 cfu per 25 ml of milk respectively for the lowest level of initial inoculum (i.e. 10⁻⁷ dilutions) and tenfold this for 10⁻⁶ dilutions. These were used to make up inoculum ratios, shown as either Cn or Wn, where C indicates Chelex extracted and cleaned samples, W indicates Wizard extracted and uncleaned samples and n indicates ratios (SE:EC:LM respectively) as follows: 1, 1∶1:1 (10⁻⁷ dilutions); 2, 1∶1:1 (10⁻⁶ dilutions); 3, 10∶1:1; 4, 1∶10:1; 5, 1∶1:10; 6, 10∶10:1; 7, 10∶1:10; 8, 1∶10:10; 9, mean average of all corresponding samples. Paired student’s two-tailed t-tests were performed between all 9 corresponding C and W datasets for both SE and LM TRF types e.g. C1 LM TRF figures were paired and compared with W1 LM TRF figures. Asterisks at the top of the corresponding W bar indicate significant differences, with one asterisk indicating a P value of <0.02, two indicating P<0.01, and three indicating P<0.0001. B: Total specific TRF detection percentages across DNA extraction and post-amplification product clean-up treatments for pre-enrichment inoculum ratio experiments as above. Specified TRFs detected for both <i>S. enterica</i> (SE) and <i>L. monocytogenes</i> (LM), and are indicated as described. Error bars shown are +/-1SD for all data, derived from triplicate datasets. Paired student’s two-tailed t-tests were performed between corresponding Clean Chelex and Wizard extracted DNA datasets for all eight TRFs. Asterisks at the top of the corresponding bar indicate significant differences, with one asterisk indicating a P value of <0.05, two indicating P<0.02, and three indicating P<0.005.</p

Strains used <i>in vitro</i> for M-TRFLP testing.

*<p>Used in milk inoculation experiments. ATCC: American Type Culture Collection, USA.; CMCC: Colworth Microbiology Culture Collection, Unilever, UK.; NCIMB and FDL: NCIMB Ltd, Aberdeen, UK.; NCTC: National Collection of Type Cultures, U.K.; SPRC and TX: Food and Drug Administration, U.S.A.; MI: This study.</p

Terminal restriction fragments (TRFs) predicted and produced from five target genes following individual species amplification and <i>Hha</i>I digestion.

<p>All TRFs are listed using the code: (dye)(length in bp).</p>*<p>TRFs unique to that species and definable on analysis.</p>a<p>TRFs combined into a single bin due to lack of distinction on analysis.</p>b<p>TRFs combined into a single bin due to lack of distinction on analysis.</p><p>UTRF: TRF derived from the upstream i.e. forward primer end of the amplicon.</p><p>DTRF: TRF derived from the downstream i.e. reverse primer end of the amplicon.</p

Multiplex Polymerase Chain Reaction amplification products shown following gel electrophoresis.

<p>Lanes 1 and 12 contain HyperLadder V (Bioline), with the remaining lanes containing products derived from 6 ng genomic DNA from as shown. Internal amplification control (IAC) template DNA was included in all reactions. Templates were amplified in isolation (apart from universal inclusion of the IAC) or in combination as described in the figure using a 15-primer multiplex PCR. Codes used are as follows: SE, <i>Salmonella enterica</i> MISE807439; LM, <i>Listeria monocytogenes</i> CMCC2993; LW, <i>Listeria welshimeri</i> CMCC3366; LG, <i>Listeria grayi</i> CMCC3362. Patterns for <i>L. seeligeri</i>, <i>L. murrayi</i>, <i>L. ivanovii</i> and <i>L. innocua</i> were identical to that of <i>L. welshimeri</i>, and all patterns shown were representative of all other strains tested of the same species. Size standards are descirbed on the left of the figure in base pairs (bp), while PCR products are described on the right of the figure with both their name and size in bp. ² indicates the <i>prs</i> amplimer from <i>Listeria grayi</i> only, with ¹ denoting the corresponding product from all other <i>Listeria</i> species. Electrophoresis was performed on a 1.7% agarose gel at 70 volts for 1.5 hrs with EtBr, with 4 ul of each product loaded.</p