Figure 1.
Multiplex Polymerase Chain Reaction amplification products shown following gel electrophoresis.
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, Salmonella enterica MISE807439; LM, Listeria monocytogenes CMCC2993; LW, Listeria welshimeri CMCC3366; LG, Listeria grayi CMCC3362. Patterns for L. seeligeri, L. murrayi, L. ivanovii and L. innocua were identical to that of L. welshimeri, 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 prs amplimer from Listeria grayi only, with 1 denoting the corresponding product from all other Listeria 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.
Table 1.
Genes targeted and oligonucleotide primers designed.
Table 2.
Terminal restriction fragments (TRFs) predicted and produced from five target genes following individual species amplification and HhaI digestion.
Figure 2.
M-TRFLP profile and analysis of pre-amplification DNA template mixes.
A: M-TRFLP profile of pre-enrichment mix of five strains of each of L. monocytogenes, S. enterica and E. coli, following Chelex extraction of DNA, multiplex PCR, post-PCR cleanup and HhaI restriction digestion. TRFs are labelled as the genes they represent and the sizes reported in base pairs, with rpoB and fusA TRFs S. enterica specific and TRFs from recA, hly and prs L. monocytogenes specific. Inocula were mixed in the ratio of 1∶1:10 in favour of Listeria, and DNA extracted from the sample post-enrichment using a Chelex-based procedure prior to PCR amplification and the products purified following PCR and prior to restriction digestion with HhaI. The TRF labelled IAC is the internal amplification control, and the IAC TRF with the asterisk undigested. B: TRF detection from two- and three-way pre-amplification template mix experiments. True and false positives and true and false negatives are indicated. Total specific TRF detection for all data is presented on the left and just two-way mix data on the right. The presence of bacteria specifically indicated by each TRF are coded as follows: SE, S. enterica; LG, L. grayi; LI, L. innocua; LV, L. ivanovii; LM, L. monocytogenes; LS, L. seeligeri; LW, L. welshimeri. *Indicates more than one target Listeria species is indicated by the presence of this TRF: L1 indicates the presence of LI, LM, LS, or LW; L2 indicates LG, LI, LV, LM, or LS; L3 indicates LI, LV, LM, or LW; L4 indicates LI, LV, or LS.
Figure 3.
Terminal Restriction Fragment (TRF) detection for DNA extraction and post-amplification clean-up protocols for inoculated milk experiments.
A: TRFs detected for both S. enterica (SE) and L. monocytogenes (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 S. enterica (SE), E. coli (EC) and L. monocytogenes (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 S. enterica (SE) and L. monocytogenes (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.
Table 3.
Strains used in vitro for M-TRFLP testing.