WO1998044153A1 - DETECTION OF LISTERIA MONOCYTOGENES, LISTERIA SPP., AND $i(RHODOCOCCUS COPROPHILUS) - Google Patents

DETECTION OF LISTERIA MONOCYTOGENES, LISTERIA SPP., AND $i(RHODOCOCCUS COPROPHILUS) Download PDF

Info

Publication number
WO1998044153A1
WO1998044153A1 PCT/NZ1998/000044 NZ9800044W WO9844153A1 WO 1998044153 A1 WO1998044153 A1 WO 1998044153A1 NZ 9800044 W NZ9800044 W NZ 9800044W WO 9844153 A1 WO9844153 A1 WO 9844153A1
Authority
WO
WIPO (PCT)
Prior art keywords
primers
primer
dna
listeria
pcr
Prior art date
Application number
PCT/NZ1998/000044
Other languages
French (fr)
Inventor
Marion Grace Savill
Rachael Elizabeth Mccormick
Original Assignee
Institute Of Environmental Science & Research Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute Of Environmental Science & Research Limited filed Critical Institute Of Environmental Science & Research Limited
Priority to AU68585/98A priority Critical patent/AU6858598A/en
Publication of WO1998044153A1 publication Critical patent/WO1998044153A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/16Primer sets for multiplex assays

Definitions

  • the invention relates to a method of detecting Listeria and Listeria monocytogenes and in particular to a PCR method for ⁇ e detection of Listeria arid Listeria monocytogenes. It also relates to primers for use in detecting Listeria and Listeria monocytogenes.
  • the invention also relates to a method of detecting Rhodococcus coprophilus.
  • the invention relates to a PCR method of detecting Rhodococcus coprophilus. It also relates to primers for use in detecting Rhodococcus coprophilus.
  • Listeria is a bacterial pathogen found in contaminated food.
  • L.monocytogenes primers have been disclosed previously (Mengaud 198S). However the specificity is low and nonspecific binding is relatively high.
  • Rhodococcus coprophilus (Gram positive to Gram variable) was recognised almost 20 years ago as a potential indicator of domesticated herbivores (Rowbotham and Cross 1977; Mara and Oragui, 1981) but traditional methods of culturing (2- weeks incubation) have limited its application. Traditional culturing procedures take up to 14 days to establish and speeding up identification would also be an advantage. Enumeration of R. coprophilus by traditional culturing methods also
  • SUBSTITUE SHEET (Rule 26) presents potential problems due to the possibility of break-up of actinomycete hyphae into coccoid elements.
  • the invention provides a primer which reacts with Listeria monocytogenes but which does not react with related or unrelated species of bacteria.
  • the primer is a DNA primer. More preferably it is targeted against the Listeriolysin O gene.
  • the primer is selected from the group comprising 31 OF, 1016R, 715F and 1183R. A combination of two primers is especially preferred.
  • the invention provides a method for detecting Listeria monocytogenes in a sample comprising the use of a primer or combination of primers above in a polymerase chain reaction (PCR) method.
  • PCR polymerase chain reaction
  • the invention also provides a primer which reacts with Listeria but which does not react with related or unrelated species of bacteria.
  • the primer is a DNA primer. More preferably it is targeted against the 23 S rRNA DNA.
  • Preferably it is selected from the group comprising L318F, L1541F, L1993F, L559R, L2038R and L2534R.
  • the invention provides a method for detecting Listeria in a sample comprising the use of a primer or combination of primers selected from the group comprising L318F, L1541F, L1993F, L559R, L2038R, and L2534R in a PCR method.
  • the invention also provides a method of detecting L.monocytogenes in a sample comprising the step of using a nucleotide primer which reacts specifically with L.monocytogenes and which does not react with related species of bacteria to detect the presence or absence of L.monocytogenes wherein two primers selected from the group comprising 31 OF, 1016R, 715F and 1183R are used together with two primers selected from the group comprising L318F, L 154 IF, L1993F, L559R, L2038R and L2534R.
  • the invention provides a primer which reacts with Rhodococcus coprophilus but which does not react with related or unrelated species of bacteria.
  • the primer is a DNA primer and more preferably it is targeted against a 16S rRNA DNA sequence.
  • the primer is selected from the group comprising 143F, 568R, 419F, 443F, 467R and l l24R.
  • the invention provides a method for detecting Rhodococcus coprophilus in a sample comprising the step of using a DNA primer which reacts specifically with R. coprophilus and which does not react with related species of bacteria to detect the presence or absence of R.coprophilus in the sample in a PCR method.
  • the primer is of a DNA primer and more preferably is targeted against a 16S rRNA DNA sequence.
  • the DNA primer is selected from the group comprising 143F, 568R, 419F, 443F, 467R and 1124R.
  • the invention provides a method of producing a primer which is able to react with R. coprophilus but which does not react with related or unrelated bacterial species.
  • the method may comprise selecting suitable, specific primers on the basis of a 16S rRNA DNA sequence alignment of R. coprophilus and related genera.
  • a suitable primer giving a specific reaction with R.coprophilus are tested against related and unrelated species.
  • the invention also provides a method of producing a primer which is able to react with L.monocytogenes but which does not react with related or unrelated bacterial species.
  • the denaturation step is preferably carried out at 92-95°C, annealing at 52-65°C and extension at 65-80°C.
  • the denaturation step is preferably carried out at 92-98°C, annealing at 60-70°C and extension at 65-80°C.
  • the method is preferably used for the detection of L.monocytogenescytogenes in a food sample.
  • the method is preferably used for the detection of R. coprophilus in a water sample.
  • Figure 1 shows R. coprophilus 16S rRNA and associated primers; R. coprophilus specific primers are shown in bold;
  • Figure 2 shows PCR using new primesr 419F-1124R and 419F-568R;
  • Figure 3 shows PCR using new primers, 143F-568R
  • Figure 4 shows primers 419Fr568R with non related genera
  • Figure 5 shows primers 143F-568R with non related genera
  • Figure 6 shows primers 143F-568R and 419F-568R with some Rhodococcus species
  • Figure 7 shows primers 143F-568R and 419F-568R with some Rhodococcus species at 60 degree Celcius;
  • Figure 8 shows the effect of temperature on Rxoprophilus and R.zopfii
  • Figure 10 shows the effect of MgC12- narrow range of concentrations
  • FIG. 11 shows the effect of primer concentration
  • Figure 12 shows a narrower range of primers
  • FIG. 13 shows the effect of DNA concentration
  • FIG 14 shows the effect of DNA concentration (lanes 9-17 only).
  • Figure 15 shows dNTP Optimisation
  • Figure 16 shows fine tuning dNTP concentration
  • Figure 18 shows 143F-568R with Rhodococcus and Rhodococcus related genera
  • Figure 19 shows DNA from extraction 2 run on a 1% agarose gel
  • Figure 20 shows Phylogenetic dendrogram based on the comparison of 16S rRNA sequences of Rhodococcus and Rhodococcus related genera
  • Figure 21 shows results of PCR with R. coprophilus DNA and different sets of primers
  • Figure 22 shows L. monocytogenes listeriolysin O gene and associated specific " primers
  • Figure 23 shows Listeria 23 S rRNA gene and associated Listeria specific primers
  • Figure 24 shows Listeria monocytogenes with all L. monocytogenes specific primers
  • Figure 25 shows positive and negative food samples with all L. monocytogenes specific primers
  • Figure 26 shows two positive food samples with all L. monocytogenes specific primer pairs
  • Figure 27 shows Listeria species against the four different primer pairs
  • Figure 28 shows Shigella flexneri, Shigella sonnei and Salmonella menston with L. monocytogenes specific primers
  • Figure 29 shows two L. monocytogenes positive food samples (298 and 297), Yersinia enterolitica, Campylobacter jejuni with L. monocytogenes specific primers;
  • Figure 30 shows the effect of BSA and DNA concentration on PCR product in food samples
  • Figure 31 shows Listeria specific primers against four Listeria species and two closely related bacteria
  • Figure 32 shows the effect of temperature on the specificity of the Listeria specific primers
  • Figure 33 shows the effect of MgCl 2 on L1541F & L2038R using L. monocytogenes and B.subtilis;
  • Figure 34 shows Multiplex (310F & 1016R, L318F & L559R);
  • Figure 35 shows Optimisation of MgCl 2 (lanes 2-9). Different muliplexes (lanes 10-12);
  • Figure 36 shows L.monocytogenes DNA extracted using the current procedure and tested using the different multiplex systems
  • Figure 37 shows the use of the new primers 31 OR and 1016R
  • Figure 38 shows a PCR using known primers
  • Figure 39 shows the use of new primers
  • Figure 40 shows the use of known primers
  • Figure 41 shows food samples using the new primers
  • Figure 42 shows food samples using known primers
  • Figure 43 shows food samples using the new primers
  • Figure 44 shows food samples using known primers
  • Figure 45 shows food samples using new primers
  • Figure 46 shows food samples using known primers
  • Figure 47 shows the names given to the Listeria species and related genera used in the phylogenetic dendrogram
  • Figure 48 shows a phylogenetic dendrogram based on the comparison of 23 S rRNA sequences of Listeria and related genera
  • Figure 49 shows the specificity of primer pairs with E. coli and B. subtilis
  • Figure 50 shows Bacillus cereus with L. monocytogenes specific primers
  • Figure 51 shows Staphlococcus aureus
  • Figure 52 shows Enterococcus faecalis (lanes 2-10) and Aeromonas hydrophila (lanes 11-19);
  • Figure 53 shows a list of the bacterial species tested.
  • Aeromonas hydrophila BHI broth 2 35 Bacillus cereus BHI broth 2 35 Bacillus subtilis BHI broth 2 35 Enterobacter aerogenes BHI broth 1 35 Enterococcus faecalis BHI broth 2 35 Escherichia coli BHI broth 1 35 Morganella morganii BHI broth 2 35 Pseudomonas aeruginosa BHI broth 2 35 Staphylococcus aureus BHI broth 2 35 Staphylococcus epidermidis BHI broth 2 35
  • BHI agar 5.3 g BHI agar in 100 mis distilled water. Autoclave 121 °C for 15 mins.
  • Both plates were washed with Jif and rinsed with dd H 2 O and then with 95% ethanol.
  • the good side of small plate was covered with repel silane and the good side of the large plate with bind silane.
  • 4mm plastic spacers were placed on the sides of the large plate and a strip of 4mm paper along the bottom. The plates were sandwiched together with gel tape.
  • the urea was prepared while the plates were taped. 42.0 g urea was added to 36 mis of dd H 2 O and warmed to dissolve. To the urea the following was added:
  • the gel was run with loading buffer in 1 x Sanger TBE for 30 mins at 1800V, 40 mA, 50W). 8 ⁇ l of sample was added to 6 ⁇ l of loading buffer and denatured for 4 minutes at 94 °C. The wells of the gel were flushed with 1 x Sanger TBE to remove urea and 8 ⁇ l of sample was added per well. The gel was soaked in ethidium bromide/Sanger TBE (200 ⁇ l in 1 litre) but the bands were too faint to visualise and the gel was silver stained instead.
  • the gel was first fixed in 2L of 10 % glacial acetic acid for 30 minutes. After 3 washes in dd H 2 O it was agitated in staining solution (2 g silver nitrate and 3 ml formaldehyde in 2 L water) for 30 minutes. The gel was placed in IL of developing solution (60 g sodium carbonate in 2 L water and chilled to 10 °C. Immediately before use 3 ml of 37% formaldehyde was added and 400 ⁇ l sodium thiosulphate 10 mg/ml) for 2-3 minutes. The developing solution was then replaced with the remaining 1 L and the gel agitated for another 2-3 minutes. It was then rinsed twice in water and dried vertically over night.
  • staining solution 2 g silver nitrate and 3 ml formaldehyde in 2 L water
  • PCR amplification was performed in 0.5 ml tubes in a total reaction volume of 100 ⁇ l using 50 mM KCl, 10 mM Tris and 2.5 mM MgCl 2 pH 8.4, 5 pmoles of each primer (0.05 ⁇ M), 2.5 Units of Taq and 200 ⁇ M of each dNTP.
  • Reverse primer 1.0 ⁇ l dNTP's (200 ⁇ M each) 0.8 ⁇ l
  • the reaction mixture was overlaid with 50 ⁇ l of nujol oil and 2 ⁇ l of DNA was added (200 ng/100 ⁇ l).
  • the tubes were then briefly centrifuged and then they were placed in a programmable DNA thermal cycler (Perkin-Elmer Thermal Cycler 480).
  • the thermal profile was 94°C denaturing for 1 min, 55°C annealing for 1 min, 72°C extension for 1 min, over 30 cycles followed by a final 8 min extension step at 72°C.
  • PCR products were analysed by gel electrophoresis using 2% agarose gels in TBE buffer.
  • the gel was placed on the light box gel side up and the film placed onto the gel glossy white (emulsion) side down and exposed for 20 sees. Grey side up the film was placed in developer until the bands appeared. It was washed in water, fixer and again in water.
  • Nucleic acid extraction 300 ⁇ l of the above phenol chloroform was added to the lysed culture and gently mix end over end for 10 mins. It was then centrifuge for 15 mins at 13,000 rpm and the top aqueous layer transferred to another tube. A further 300 ⁇ l of phenol chloroform was added and mixed end over end for 10 mins. The extract was then centrifuged as before and the top aqueous phase transferred to a new tube. 300 ⁇ l of chloroform was added and mixed end over end for 10 mins. It was centrifuge as before, the top aqueous phase transferred to a new tube and 25 ⁇ l of 3M sodium acetate pH 5.2 was added with 600 ⁇ l of absolute ethanol.
  • Tubes were then stored either overnight at -20°C or for at least 1 hour at -70°C.
  • the tubes were centrifuged again at 13,000 rpm for 15 mins and the supernatant discarded. 600 ⁇ l of 70 % ethanol was added and centrifuged again as before. As much as possible of the supernatant was removed with a pipette and any remaining ethanol evaporated by placing the tube on a 100°C hot block until the tube was dry.
  • the DNA was then resuspended in 20 ⁇ l of dd H 2 O and stored at -20°C.
  • a working solution was prepared by dilution of the stock to 100 ng/ml. 2 ⁇ l of this working solution was added to each PCR assay to give a final concentration of 200 ng/100 ⁇ l. If the DNA was more dilute than this it was used neat in the PCR reaction, 2 ⁇ l being added.
  • primers were chosen, 4 selected to be specific for R. coprophilus and another four which were specific for all bacteria to be used to determine which of the R. coprophilus specific primers were working.
  • Figure 1 shows R. coprophilus 16S rRNA and associated primers.
  • ddH O was added to the primers to give a stock concentration of 100 nmoles/ml. A further 1/20 dilution of each primer was done to produce a working solution for PCR of 5 pmoles/ ⁇ l.
  • the first step was to test the different combinations of primers with R. coprophilus to determine that bands would be obtained.
  • R. coprophilus was grown as stated in 'Bacteria and Cultivation' and the DNA extracted as in 'DNA extraction' extraction 1.
  • the PCR was carried out according to 'PCR assay' above. The procedure was then repeated for all the other species outlined in Table 9, using the DNA from extraction 2. All PCR products were detected using agarose gels and visualised using ethidium bromide as outlined in 'Detection of PCR products'. The results are summarised below.
  • primer 143F can distinguish between R. coprophilus and all the bacteria tested including the closely related R. equi, by the absence of a band whether it is used with or without a specific primer. From the DNA sequence alignment, for 143F to be able to distinguish between R. coprophilus and R. equi it must be able to pick up a difference of 3 bp on the 3' end.
  • 143F If 143F is to be used it must be capable of picking up smaller differences such as those shown above for R. marinonascens and R. fascians. Both these cultures were ordered as these should be some of the most difficult to differentiate.
  • Primer 467R can only be used in conjunction with 143F and as 143F to date has always worked it can't be determined if 467F is working or not. Therefore another non specific forward primer was ordered, 27F, to test whether it is working.
  • Primer 443 F is unable to distinguish R. coprophilus from other species and therefore 143F is preferred as the forward primer. Another forward primer was ordered to replace it, 419F.
  • Primer 1124R can't always distinguish between R. coprophilus and other species although it often produces several bands which could be a way of distinguishing or it may be that the bands it does produce are of different lengths. All the PCR products from 1124R and 443F were run on a 4% acrylamide gel to determine if there is any difference in band size. From this it was determined that no significant difference in band size could be seen and that 1124R would not be suitable for use as a primer. A replacement primer for 1124R was ordered, 568R. Table 10. Details of new primers
  • the new primers were diluted to give a stock concentration of 100 nmoles/ml. A further 1/20 dilution was carried out to give a working solution of 5 pmoles/ ⁇ l.
  • PCR amplification was performed as outlined in 'PCR assay' with the following exception.
  • the thermal profile on the Perkin-Elmer was: 94°C denaturing for 1 min, 60°C annealing for 1 min, 72°C extension for 1 min, over 30 cycles followed by a final 8 min extension step at 72°C.
  • Figure 2 shows PCR using new primers, 419F - 1124R and 419F - 568R.
  • Figure 3 shows PCR using new primers, 143F - 568R.
  • primer dimer Large amounts of primer dimer are seen in 419F-1124R. No primer dimer is seen with 419F-568R or 143F-568R. The latter two primers show good strong bands with R. coprophilus but not with any other of the related families tested. As 419F-1124R gave bands with N. brasiliensis work was continued with the other two sets of primers(143F-568R and 419F-568R).
  • the next step was to test the two new sets of primers with the non related species.
  • a PCR was set up for both sets of primers using the same conditions as for the last experiment.
  • Figure 4 shows Primers 419F-568R with non related genera.
  • Figure 5 shows Primersl43F-568R with non related genera.
  • Both sets of the primers perform well with all non related species.
  • the only band formed was one with B. cereus with 419F-568R, which was of a different size and easily distinguishable from the R. coprophilus band.
  • R . coprophilus DNA was also tested in a multiplex of 143F, 419F and 568R which gave two strong bands at 425 and 149 base pairs (lane 14).
  • the next step of the assay is to test both sets of primers with closely related Rhodococcus species to determine if there is any non specific bands.
  • 143F -568R showed a strong band with R.coprophilus and only a faint band with R. zopfii all other species tested showed a negative reaction.
  • 419F-568R showed a strong band with R. coprophilus but weak bands with all others tested. It was thought that these weak bands may be removed by increasing the temperature even further to 65°C or by changing the MgCl 2 concentration. Temperature was thought to be the main effector and so the experiment was repeated exactly as above except at an annealing temperature of 65°C and not 60°C.
  • Figure 7 shows Primers 143F - 568R and 419F - 568R with some Rhodococcus species at 60 °C.
  • PCR's for R. coprophilus and R. zopfii were carried out at annealing temperatures of 61°C, 62°C, 63 °C and 64°C. Apart from the annealing temperature the conditions were identical to the previous experiments. (PCR products for 60°C and 65°C from the previous experiment were run on the gel also)
  • Figure 8 shows the effect of temperature on R. coprophilus and R. zopfii.
  • the R. coprophilus band starts decreasing in intensity at 64°C so 63 °C would be optimum, but there is possibly a faint band of R. zopfii at this temperature. To allow for changes in temperature on other machines and for the possibility of other Rhodococcus species having a stronger band the temperature is to be kept at 65°C.
  • lOx PCR buffer with no MgCl was prepared (500 mM KCl, 100 mM Tris) and autoclaved. 25 mM MgCl was also prepared and autoclaved. A range of MgCl concentrations was tried around the concentration already being used.
  • Figure 9 shows the effect of MgCl 2 - Wide range of concentrations. A narrower range was then tried at 2.0, 2.2, 2.4, 2.6, 2.8 & 3.0 mM
  • Figure 10 shows the effect of MgCl 2 - Narrow range of concentrations.
  • the primer concentration used throughout the experiments was 5 pmoles/ 100 ⁇ l. To optimise the primers a range was tried of 2, 4, 5, 6, 8 & 10 pmoles/100 ⁇ l.
  • Figure 12 shows a narrower range of primers.
  • Figure 13 shows the effect of DNA concentration.
  • FIG 14 shows the effect of DNA concentration (lanes 9-17 only)
  • the usual dNTP concentration of 25mM (final cone, of 200 ⁇ M/100 ⁇ l) was further diluted 1/10 to allow a range of concentrations to be tried from 50, 100, 150, 200, 250, 300 ⁇ M.
  • Figure 15 shows dNTP Optimisation.
  • Figure 16 shows fine tuning dNTP concentration.
  • Taq is currently used at 2.5 Units/100 ⁇ l and therefore a range was tried around this at, 3, 2.5, 2.0, 1.5, 1.0 and 0.5 Units.
  • Figure 17 shows Taq Optimisation.
  • PCR amplification was performed in 0.5 ml tubes in a total reaction volume of 100 ⁇ l using 50 mM KCl, 10 mM Tris and 2.5 mM MgCl 2 pH 8.4, 5 pmoles of each primer (0.05 ⁇ M), 2.5 Units of Taq and 150 ⁇ M of each dNTP.
  • Reverse primer (5 pmoles/ ⁇ l) 1.0 ⁇ l dNTP's (25mM each) 0.6 ⁇ l
  • the reaction mixture was overlaid with 50 ⁇ l of nujol oil and 2 ⁇ l of DNA was added (200 ng/100 ⁇ l).
  • the tubes were then briefly centrifuged and then they were placed in a programmable DNA thermal cycler (Perkin-Elmer Thermal Cycler 480).
  • the thermal profile was 94°C denaturing for 1 min, 65°C annealing for 1 min, 72°C extension for 1 min, over 30 cycles followed by a final 8 min extension step at 72°C.
  • Rhodococcus and Rhodococcus related species were run including R. fascians and R. marinonascens that had not been tested before.
  • the related genera were chosen from dendrogram family trees based on the 16S rRNA homology and similarly the Rhodococcus species were chosen in the same manner.
  • the species chosen were ones that were either very closely related in DNA homology to R. coprophilus or selected species or genera indicative of other branches from the dendrogram.
  • the extreme genera (little or no homology) were also tested. Obviously not all genera or species were tested, however we believe we have selectively chosen a good representation of the field and have covered the species/genera most likely to cause false positives (most closely related).
  • An example of a genera not tested was Gordona terrae (since renamed R. terrae) however the DNA homology of this genus and R.
  • R. coprophilus at site of 143F GGGTCTAATACCGGATATGACCAT
  • R. terrae GGGTCTAATACCGGATATGACCAA
  • R. zopfii GGGTCTAATACCGGATATGACCAA
  • Rhodococcus related genera are shown below with the DNA sequences at the priming sites used (143F and 568R):
  • # Denotes R. coprophilus and the DNA sequence for which this method has been designed at the primer sites.
  • N. transvaliensis only has one mismatch on the DNA sequence at the 143F primer site, however within the DNA sequence in the 568R primer region many mismatches occur and for this reason we do not believe amplification is possible.
  • Other bacteria such as N. calcarea and N. corynebacteroides have few mismatches in the 568R primer region but more in the 143F region.
  • Gordona rubropertinctus is the most similar to R. coprophilus. However all of the genera have more mismatches than R. zopfii and therefore highly unlikely to amplify.
  • Figure 18 shows 143F-568R with Rhodococcus and Rhodococcus related genera
  • Figure 19 shows DNA extraction 2 run on a 1% agarose gel
  • Figure 20 shows phylogenetic dendrogram based on the comparison of 16S rRNA sequences of Rhodococcus and Rhodococcus related genera.
  • Figures 21 shows the results of the PCRs with Rhodococcus coprophilus DNA and different sets of primers.
  • BHI agar 5.3 g BHI agar in 100 mis distilled water. Autoclave 121 °C for 15 mins.
  • BHI broth 3.8 g BHI broth in 100 mis distilled water. Autoclave 121 °C for 15 mins.
  • Tryptic soy agar 4 g in 100 ml distilled water . Autoclave 121°C for 15 mins.
  • Tubes were then stored either overnight at -20°C or for at least 1 hour at -70°C.
  • the tubes were centrifuged again at 13,000 rpm for 15 mins and the supernatant discarded. 600 ⁇ l of 70 % ethanol was added and centrifuged again as before. As much as possible of the supernatant was removed with a pipette and any remaining ethanol evaporated by placing the tube on a 100°C hot block until the tube was dry.
  • the DNA was then resuspended in 20 ⁇ l of dd H 2 O and stored at -20°C.
  • a working solution was prepared by dilution of the stock to 100 ng/ml. 2 ⁇ l of this working solution was added to each PCR assay to give a final concentration of 200 ng/100 ⁇ l. If the DNA was more dilute than this it was used neat in the PCR reaction, 2 ⁇ l being added.
  • Listeria monocytogenes specific primers For the L. monocytogenes specific primers the listeriolysin O gene was chosen as it is only found in haemolytic bacteria and would narrow down the amount of organisms that the primers would cross react with.
  • Four primers were chosen that were thought to be specific for L. monocytogenes. Two other primer sequences, LF and LR that have already been published (Bansal 1996) were also selected. Details of each primer are given in Table 6.
  • Figure 22 shows L.monocytogenes listeriolysin O gene and associated specific primers.
  • the primer positions are as follows: the 5 ' end of 31 OF starts on base pair 310, the 5' end of 715F starts on base pair 715, the 5 ' end of 1016R complement starts on base pair 1016, the 5 ' end of 1183R complement starts on base pair 1183.
  • the 16S rRNA was compared from a large number of bacteria but there were very few regions that were specific to Listeria. Having collated all the 23 S rRNA sequence data possible from Genbank the sequences were aligned using the GCG sequence alignment package. From the alignment, areas specific to Listeria could be chosen and PCR primers designed around these areas.
  • Figure 23 shows Listeria 23 S rRNA gene and associated Listeria specific primers.
  • the primer positions are as follows:
  • the 5 ' end of L318F starts on base pair 318
  • the 5' end of L1541F starts on base pair 1541
  • the 5' end of L1993F complement starts on base pair 1993
  • the 5' end of L559R complement starts on base pair 559
  • the 5' end of L2038R complement starts on base pair 2038
  • the 5' end of L2534R complement starts on base pair 2534.
  • PCR amplification was performed in 0.5 ml tubes in a total reaction volume of 100 ⁇ l using 50 mM KCl, 10 mM Tris and 2.5 mM MgCl 2 pH 8.4, 5 pmoles of each primer (0.05 ⁇ M), 2.5 Units of Taq and 200 ⁇ M of each dNTP.
  • Reverse primer 1.0 ⁇ l dNTP's (200 ⁇ M each) 0.8 ⁇ l
  • the reaction mixture was overlaid with 50 ⁇ l of nujol oil and 2 ⁇ l of DNA was added (200 ng/100 ⁇ l).
  • the tubes were then briefly centrifuged and then they were placed in a programmable DNA thermal cycler (Hybaid Omnigene).
  • the thermal profile was 95°C denaturing for 1 min, 55°C annealing for 1 min, 72°C extension for 1 min, over 30 cycles followed by a final 8 min extension step at 72°C.
  • SUBSTITUE SHEET (Rule 26) PCR products were analysed by gel electrophoresis using 2% agarose gels in TBE buffer.
  • L. monocytogenes was grown as stated in 'Bacteria and Cultivation' and the DNA extracted as in 'DNA extraction' extraction 1. The PCR was carried out according to 'PCR assay' above.
  • Figure 24 shows Listeria monocytogenes with all L.monocytogenes specific primers.
  • Figure 25 shows Positive and negative food samples with all L. monocytogenes specific primers.
  • Figure 26 shows two positive food samples with all L. monocytogenes specific primer pairs.
  • Figure 27 shows Listeria species against the four different primer pairs.
  • Figure 28 shows Shigella flexneri, Shigella sonnei and Salmonella menston with L. monocytogenes specific primers.
  • Figure 29 shows two L. monocytogenes positive food samples (298 & 291), Yersinia enterolitica, Campylobacter jejuni with L. monocytogenes specific primers
  • Figure 30 shows Effect of BSA and DNA concetration on PCR product in food samples
  • the first step was to test the three sets of Listeria specificprimer pairs aginst the four Listeria species and two closely related bacteria, B. subtilis and S aureus.
  • Figure 31 shows Listeria specific primers against four Listeria species and two closely related bacteria.
  • the first primer pair L318F & L559R gave bands with all the Listeria species (lanes 2, 5, 8 and 11) and no bands with the other two related species B. subtilis(lane 14) and S. ⁇ ureus( ⁇ ane 17).
  • the second primer pair L 154 IF & L2038R gave a false positive with B. subtilis(lane 15) but not with S. ⁇ ureus( ⁇ ane 18).
  • the final pair, L1993F & L2534R gave false positives with both related bacteria(lanes 16 & 19). It was thought that an increase in the annealing temperature from 55°C to 62°C may improve the specificity.
  • L318F & L559R Listeria specific primers picks only the Listeria strains and could be used in the PCR assay.
  • L 154 IF & L2038R could potentially be improved with a change in the MgCl 2 concentration.
  • a PCR was set up using lOx PCR buffer with no MgCl 2
  • a 25 mM solution of Mg Cl 2 was prepared and a range of concentrations set up between 0.5 mM and 5.0 mM.
  • Two organisms were tested L. monocytogenes and B subtilis, a concentration was looked for that would produce a band with L. monocytogenes but not with B. subtilis.
  • the PCR was run at an annealing temperature of 62°C.
  • Figure 33 shows the effect of MgCl 2 on L1541F & L2038R using L.monocytogenes and B. ubtilis.
  • SUBSTITUE SHEET (Rule 26) 2.0 mM MgCl 2 concentration as a back up for L318F & L559R.
  • the B. subtilis 23 S rRNA was retrieved from the Genbank database and the 3 sets of primers compared to it using Oligo.lt was found that:
  • L2038R had 24 of its 25 bp matching the B. subtilis DNA. This would indicate that they are quite likely to form a PCR product.
  • Figure 34 shows multiplex (310F & 1016R, L318F & L559R)
  • the primer pairs worked as expected. They showed the first six samples to be L. monocytogenes positive. The seventh sample (lane 8) was a negative food sample and gave no bands on the gel. The next three were Listeria species and only the expected Listeria band formed.. The last seven organisms were not related and no PCR product was obtained. In lane 5 the bands were quite weak. The gel was rerun to see if it was a loading problem.The gel was run as before but at 70V.
  • the next step was to determine the optimum concentration of MgCl 2 for the multiplex.
  • a range of MgCl 2 concentrations was tried between 0.5 and 4.0 mM for primers 310F, 1016R, L318F and L559R (lanes 2-9) Other multiplex combinations were also tried (lanes 10-12):
  • Figure 35 shows optimisation of MgCl 2 (lanes 2-9). Different multiplexes (lanes 10-12)
  • Figure 36 shows L. monocytogenes DNA extracted using the current procedure and tested using the different mulitplex systems.
  • Viable cells resuspended and diluted in water (7.5) denatured (7.6) centrifuged (7.7) - sample PCR'd(A)
  • Figure 37 shows a procedure using primers 31 OF and 1016R.
  • DNA can be extracted from frozen cells and still give a reliable positive result.
  • Heat denatured DNA is able to be frozen and reused with no adverse effect on the results, i.e.the expected bands still amplify and there are no non specific bands.
  • the results from the new primers were then compared to the results from the known primers using a PCR procedure.
  • Figure 38 shows the results of a PCR using known primers
  • the known Listeria primers gives weak amplification products when the sample is at
  • the upper band (241 bp) is the Listeria band and the lower (706 bp) is the L. monocytogenes.
  • the upper band in sample 1 is the L. monocytogenes band and the lower one the Listeria band.
  • Figure 39 shows a method using new primers.
  • Figure 40 shows a method using known primers.
  • Figure 41 shows food samples using the new primers.
  • Figure 42 shows food samples using the known primers.
  • Figure 43 shows food samples using the primers according to the invention.
  • Figure 44 shows food samples using known primers.
  • Figure 45 shows food samples using new primers.
  • PCR amplification was performed in 0.5 ml tubes in a total reaction volume of 100 ⁇ l (This can be scaled down to 20 ⁇ l to economise on materials.) using 50 mM KCl, 10 mM Tris and 2.5 mM MgCl 2 pH 8.4, 5 pmoles of each primer (0.05 ⁇ M), 2.5 Units of Taq and 200 ⁇ M of each dNTP. It has also been determined that the addition of BSA at a final concentration of 0.2 mg/ml helps to prevent inhibiton of amplification by any contaminants in the samples. The annealing temperature has also been raised from 55°C to 62°C to increase the specificty of the assay.
  • Reverse primer L559R 1.0 ⁇ l dNTP's (200 ⁇ M each) 0.8 ⁇ l
  • the reaction mixture was overlaid with 50 ⁇ l of nujol oil and 2 ⁇ l of DNA was added (200 ng/100 ⁇ l).
  • the tubes were then briefly centrifuged and then they were placed in a programmable DNA thermal cycler (Hybaid Omnigene).
  • the thermal profile was 95 °C denaturing for 1 min, 62°C annealing for 1 min, 72°C extension for 1 min, over 30 cycles followed by a final 8 min extension step at 72°C.
  • primer Ul was found (region 528 -545) not to be Listeria specific but a universal primer and will bind to any bacteria.
  • UII the second primer (region 1566- 1587) was found to be Listeria specific on the last base pair on the 3' end with a few other mismatches throughout the sequence.
  • the 3' end of a primer is the most important region for specificity to occur. The significance of only one mismatch on the 3' end is, the assay may have great difficulty in distinguishing between Listeria and other species particularly at the low annealing temperature used (49°C) and the higher magnesium concentration used (3 mM) both of which would promote non specific binding.
  • the listeriolysin gene which codes for the protein causing haemolysis was chosen as the region of DNA specific for the L. monocytogenes species DNA sequences were taken from Genbank for all lysin genes. We obtained sequences from 5 L. monocytogenes species, L. ivanovii and L. seeligeri and using the GCG package aligned these to examine for potential primer sites. We chose four sites and in addition included the two new sites used by Dr Bansal, LF and LR (Bansal 1996 in press). All sites (9 combinations) amplified with no cross reactivity occuring with other Listeria species. However we chose one combination based on the strength and length of the product from primers 31 OF and 1016R.
  • Streptococcus canis Vibrio par ahaemolyticus Streptococcus equisimilis
  • the DNA was checked against the new primers (31 OF and 1016R) using the DNA program Oligo. All the L. monocytogenes species showed complete binding of both primers and all produced a 706 base pair fragment. All the other species and genera tested showed no or only an extremely small degree of potential primer binding. Theoretically the chosen primers should not bind with this DNA and certainly will not cause ambiguity of the results.
  • L. monocytogenes primers LMI and LMII
  • LMI and LMII L. monocytogenes primers
  • the new L.monocytogenes primers 31 OF and 1016R are in the same area as LMI and LMII but are upstream.
  • the 31 OF primer is three bases and the 1016R primer is twelve bases upstream. Both these differences incorporate more mismatches at the 3' ends and along the whole template.
  • the primers are 25 bases not 18 bases in length. The effects of both are to increase specificity of binding to the L. monocytogenes species and obviously reducing the non specific binding observed.
  • a known DNA method based on earlier work of Dr Bansal used the Listeria specific primers of Ul and UII combined with the L. monocytogenes primers LMI and LMII. This combination of primers has been found to cause many non specific bands.
  • the Listeria primers Ul and UII (UII is also known as LII in Bansal 1996) have been combined with L. monocytogenes primers LF and LR. These latter two primers are used at a lower annealing temperature of 51°C.
  • the LR primer has no L. monocytogenes sites on the last three bases at the 3' end and the LF primer has only one specific site on the 3' end.
  • the LF primer has therefore fewer specific sites on this 3' end than our corresponding primer and is potentially more likely to mistype than the primers according to the present invention.
  • the primers according to the present invention have been designed to maximise the nucleotide differences between all the existing Listeria sequences. Preexisting primers do not. The present primers are superior and significantly different to any known primers.
  • the new PCR has been tested against three closely related bacteria and a number of other unrelated organisms.
  • the extreme genera (little or no homology) were tested particularly if they were food related pathogens that may be present in the types of samples for which the PCR will ultimately be applied.
  • a larger field of related organisms are still to be tested (see future work)
  • Figure 53 shows a list of bacterial species tested to date.
  • This current PCR method is based on amplification of certain sequences of DNA on the Listeria monocytogenes genome. It is a multiplex method where two pairs of primers are used in one PCR reaction. One pair of primers is designed specifically for genus Listeria identification and another pair for species monocytogenes identification.
  • DNA is extracted from the bacterial cells by heat blasting the cells and adding aliquots to the PCR reaction.
  • Listeria is a contaminant of food samples and is pathogenic to humans.
  • the new, specific primers will enable the detection of Listeria and L.monocytogenes in food samples.
  • Rcoprophilus is a contaminant of water and is also pathogenic to humans.
  • the invention provides new, specific primers allowing for a simple and convenient assay for its detection. This would enable one to determine whether a sample is polluted with faecal material.
  • Rhodococcus coprophilus An aerobic nocardioform actinomycete belonging to the "rhodocrous": complex. Journal of General Microbiology 100: 123- 138.

Abstract

The invention relates to methods of detecting the: genus Listeria; species Listeria monocytogenes; and species Rhodococcus coprophilus. All primers that react with the named species, but do not cross react with related or unrelated species of bacteria are claimed. The preferred primers for the: genus species Listeria monocytogenes; Listeria; and species Rhodococcus coprophilus; come from the: Listeriolysin O (HylA); 23s rRNA subunit; and 16s rRNA, genes respectively.

Description

DETECTION OF LISTERIA MONOCYTOGENES, LISTERIA SPP., AND RHODOCOCCUS COPROPHILUS
TECHNICS FIELD
The invention relates to a method of detecting Listeria and Listeria monocytogenes and in particular to a PCR method for ±e detection of Listeria arid Listeria monocytogenes. It also relates to primers for use in detecting Listeria and Listeria monocytogenes.
The invention also relates to a method of detecting Rhodococcus coprophilus. In particular the invention relates to a PCR method of detecting Rhodococcus coprophilus. It also relates to primers for use in detecting Rhodococcus coprophilus.
BACKGROUND .ART
Listeria is a bacterial pathogen found in contaminated food.
Current microbiological culture procedures for the detection of Listeria are labourious and time consuming. Many authors in the literature have laboured this point. The recent developments in molecular biology have raised the possibility of detecting pathogens in foods and other samples. For this reason we decided to investigate the use of DNA techniques for the detection of Listeria monocytogenes.
A DNA method used by the Australian Molecular Microbiological Laboratory, Division of Analytical Laboratories, NSW Health Department, Lidcombe, Sydney has been found to produce too many non specific products causing a problem in positive identification of L.monocytogenes. For this reason we decided to go back to basics using DNA sequences from Gεnbank and designing our own primers and establish our own PCR system.
Some L.monocytogenes primers have been disclosed previously (Mengaud 198S). However the specificity is low and nonspecific binding is relatively high.
The development of a simple and convenient assay for Listeria monocytogenes would be useful. It would, for example, enable one skilled in the art to determine whether a sample, for example, a food sample, was contaminated with L.monocytogenes. Contamination of New Zealand's aquatic environments by faecal material degrades them for use as a drinking supply or for recreation because of the potential presence of pathogenic microbes. New Zealand with its high animal to human ratio would be expected to have a high propoπion of animal faecal material in rural waterways. The main international opinion is that human faecal pollution constitutes a greater disease risk than animal pollution, however this has not been conclusively established. Whatever their relative quantities and risk factors, managemant of human effluents is generally accorded a higher priority than animal, since they are more likely to contain human pathogens than animal faeces. Current methods used for faecal indication do not allow the sources of contamination to be differentiated.
Rhodococcus coprophilus (Gram positive to Gram variable) was recognised almost 20 years ago as a potential indicator of domesticated herbivores (Rowbotham and Cross 1977; Mara and Oragui, 1981) but traditional methods of culturing (2- weeks incubation) have limited its application. Traditional culturing procedures take up to 14 days to establish and speeding up identification would also be an advantage. Enumeration of R. coprophilus by traditional culturing methods also
SUBSTITUE SHEET (Rule 26) presents potential problems due to the possibility of break-up of actinomycete hyphae into coccoid elements.
Initial searches on databases for sequence data yielded very little information on Rhodococcus species. Some sequence data has been entered into the Genbank database by F. A. Rainey et al. 1995. Complete sequences for many Rhodococcus species (F. A. Rainey et al. 1995) and Rhodococcus related species (F. A. Rainey et al. 1995) are available. The part of the DNA targeted by the primers is the 16S rRNA DNA which has shown promise in its ability to differentiate at a genus and species level (Kreader 1995). Since the 16S rRNA DNA has multiple copies, theoretically fewer cells should be required for initial PCR reactions. As there is much information published on the 16S rRNA already it enables a large variety of organisms to be compared which makes selecting a R. coprophilus specific area of the DNA for primer annealing much simpler.
The development of a simple and convenient assay for R. coprophilus would be useful and would, for example, enable one skilled in the art to determine whether a sample such as a water sample was polluted with faecal material animal in origin.
Accordingly, it is an object of this invention to go at least some way in overcoming problems with known methods of detecting L.monocytogenes or R. coprophilus and to provide an assay for the detection of L.monocytogenes and to provide an assay for the detection of R.coprophilus, or to at least provide the public with a useful choice.
DISCLOSURE OF THE INVENTION
The invention provides a primer which reacts with Listeria monocytogenes but which does not react with related or unrelated species of bacteria.
Preferably the primer is a DNA primer. More preferably it is targeted against the Listeriolysin O gene.
Preferably the primer is selected from the group comprising 31 OF, 1016R, 715F and 1183R. A combination of two primers is especially preferred.
In another aspect the invention provides a method for detecting Listeria monocytogenes in a sample comprising the use of a primer or combination of primers above in a polymerase chain reaction (PCR) method.
The invention also provides a primer which reacts with Listeria but which does not react with related or unrelated species of bacteria.
Preferably the primer is a DNA primer. More preferably it is targeted against the 23 S rRNA DNA.
Preferably it is selected from the group comprising L318F, L1541F, L1993F, L559R, L2038R and L2534R.
Any combination of two of the primers selected from the group comprising L318F, L1541F, L1993F, L559R, L2038R, and L2534R is preferred. In another aspect the invention provides a method for detecting Listeria in a sample comprising the use of a primer or combination of primers selected from the group comprising L318F, L1541F, L1993F, L559R, L2038R, and L2534R in a PCR method.
The invention also provides a method of detecting L.monocytogenes in a sample comprising the step of using a nucleotide primer which reacts specifically with L.monocytogenes and which does not react with related species of bacteria to detect the presence or absence of L.monocytogenes wherein two primers selected from the group comprising 31 OF, 1016R, 715F and 1183R are used together with two primers selected from the group comprising L318F, L 154 IF, L1993F, L559R, L2038R and L2534R.
In another aspect, the invention provides a primer which reacts with Rhodococcus coprophilus but which does not react with related or unrelated species of bacteria.
Preferably the primer is a DNA primer and more preferably it is targeted against a 16S rRNA DNA sequence.
More preferrably the primer is selected from the group comprising 143F, 568R, 419F, 443F, 467R and l l24R.
In another aspect, the invention provides a method for detecting Rhodococcus coprophilus in a sample comprising the step of using a DNA primer which reacts specifically with R. coprophilus and which does not react with related species of bacteria to detect the presence or absence of R.coprophilus in the sample in a PCR method.
Preferably the primer is of a DNA primer and more preferably is targeted against a 16S rRNA DNA sequence. Preferably the DNA primer is selected from the group comprising 143F, 568R, 419F, 443F, 467R and 1124R.
In a further aspect, the invention provides a method of producing a primer which is able to react with R. coprophilus but which does not react with related or unrelated bacterial species.
Preferably the method may comprise selecting suitable, specific primers on the basis of a 16S rRNA DNA sequence alignment of R. coprophilus and related genera. Preferably a suitable primer giving a specific reaction with R.coprophilus are tested against related and unrelated species.
The invention also provides a method of producing a primer which is able to react with L.monocytogenes but which does not react with related or unrelated bacterial species.
In the method for detecting Listeria monocytogenes the denaturation step is preferably carried out at 92-95°C, annealing at 52-65°C and extension at 65-80°C.
In the methods for detecting Listeria and Rhodococcus coprophilus the denaturation step is preferably carried out at 92-98°C, annealing at 60-70°C and extension at 65-80°C.
With respect to L.monocytogenes the method is preferably used for the detection of L.monocytogenescytogenes in a food sample. With respect to R. coprophilus, the method is preferably used for the detection of R. coprophilus in a water sample. BRIEF DESCRIPTION OF DRAWINGS
Embodiments of the invention will now be described, by way of example only, with reference to the drawings, in which:
Figure 1 shows R. coprophilus 16S rRNA and associated primers; R. coprophilus specific primers are shown in bold;
Figure 2 shows PCR using new primesr 419F-1124R and 419F-568R;
Figure 3 shows PCR using new primers, 143F-568R;
Figure 4 shows primers 419Fr568R with non related genera;
Figure 5 shows primers 143F-568R with non related genera;
Figure 6 shows primers 143F-568R and 419F-568R with some Rhodococcus species;
Figure 7 shows primers 143F-568R and 419F-568R with some Rhodococcus species at 60 degree Celcius;
Figure 8 shows the effect of temperature on Rxoprophilus and R.zopfii;
Figure 9 shows the effect of MgC12- wide range of concentrations;
Figure 10 shows the effect of MgC12- narrow range of concentrations;
Figure 11 shows the effect of primer concentration;
Figure 12 shows a narrower range of primers;
Figure 13 shows the effect of DNA concentration;
Figure 14 shows the effect of DNA concentration (lanes 9-17 only);
Figure 15 shows dNTP Optimisation;
Figure 16 shows fine tuning dNTP concentration;
Figure 17 shows Taq Optimisation
Figure 18 shows 143F-568R with Rhodococcus and Rhodococcus related genera;
Figure 19 shows DNA from extraction 2 run on a 1% agarose gel;
Figure 20 shows Phylogenetic dendrogram based on the comparison of 16S rRNA sequences of Rhodococcus and Rhodococcus related genera;
Figure 21 shows results of PCR with R. coprophilus DNA and different sets of primers; Figure 22 shows L. monocytogenes listeriolysin O gene and associated specific "primers;
Figure 23 shows Listeria 23 S rRNA gene and associated Listeria specific primers;
Figure 24 shows Listeria monocytogenes with all L. monocytogenes specific primers;
Figure 25 shows positive and negative food samples with all L. monocytogenes specific primers;
Figure 26 shows two positive food samples with all L. monocytogenes specific primer pairs;
Figure 27 shows Listeria species against the four different primer pairs;
Figure 28 shows Shigella flexneri, Shigella sonnei and Salmonella menston with L. monocytogenes specific primers;
Figure 29 shows two L. monocytogenes positive food samples (298 and 297), Yersinia enterolitica, Campylobacter jejuni with L. monocytogenes specific primers;
Figure 30 shows the effect of BSA and DNA concentration on PCR product in food samples;
Figure 31 shows Listeria specific primers against four Listeria species and two closely related bacteria;
Figure 32 shows the effect of temperature on the specificity of the Listeria specific primers;
Figure 33 shows the effect of MgCl2 on L1541F & L2038R using L. monocytogenes and B.subtilis;
Figure 34 shows Multiplex (310F & 1016R, L318F & L559R);
Figure 35 shows Optimisation of MgCl2 (lanes 2-9). Different muliplexes (lanes 10-12);
Figure 36 shows L.monocytogenes DNA extracted using the current procedure and tested using the different multiplex systems;
Figure 37 shows the use of the new primers 31 OR and 1016R;
Figure 38 shows a PCR using known primers;
Figure 39 shows the use of new primers;
Figure 40 shows the use of known primers;
Figure 41 shows food samples using the new primers;
Figure 42 shows food samples using known primers;
Figure 43 shows food samples using the new primers; Figure 44 shows food samples using known primers;
Figure 45 shows food samples using new primers;
Figure 46 shows food samples using known primers;
Figure 47 shows the names given to the Listeria species and related genera used in the phylogenetic dendrogram;
Figure 48 shows a phylogenetic dendrogram based on the comparison of 23 S rRNA sequences of Listeria and related genera;
Figure 49 shows the specificity of primer pairs with E. coli and B. subtilis;
Figure 50 shows Bacillus cereus with L. monocytogenes specific primers;
Figure 51 shows Staphlococcus aureus;
Figure 52 shows Enterococcus faecalis (lanes 2-10) and Aeromonas hydrophila (lanes 11-19);
Figure 53 shows a list of the bacterial species tested.
MODES OF CARRYING OUT THE INVENTION
A) Development of a PCR method for the detection of Rhodococcus coprophilus
Experimental
Bacterial Strains & Cultivation
The bacterial strains included in this specification are listed in Table 1.
Table 1
Bacterial Strain Source
Rhodococcus Species
R. coprophilus ATCC 29080, NCTC 10994, DSM 43347T
R. equi ATCC 6939, NCTC 1621, DSM 20307
R. fascians ATCC 12974, DSM 20669
R. marinonascens DSM 43752 T
R. rhodnii DSM 43959
R. rhodochrous ATCC 13808, NCTC 10210, DSM 43241 T
R. rhodochrous ATCC 271, DSM 43274
R. ruber DSM 43338 T
R. zopfli ATCC 51349 T
Rhodococcus Related Species
Actinomyces naeslundii ATCC 12104, NCTC 10301 Corynebacterium xerosis ATCC 373 Gordona bronchialis NZ isolate MY 89/0484 Nocardia brasiliensis ATCC 19295, NCTC 10300 Streptomyces griseus NCTC 7807
Other Bacteria
Aeromonas hydrophila ATCC 7966, NCTC 8049, NCIB 9240 Bacillus cereus ATCC 10702, NCTC 8035, NCIB 8122 Bacillus subtilis ATCC 6051, NCTC 3610, NCIB 3610 Enterobacter aerogenes ATCC 13048, NCTC 10006 Enterococcus faecalis ATCC 19433, NCTC 775, NCDO 581 Escherichia coli ATCC 25922 Morganella morganii ATCC 25830, NCTC 235 Pseudomonas aeruginosa ATCC 25668, NCTC 10662 Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis ATCC 12228 ATCC American Type Culture Collection. NCTC National Collection of Type Cultures. NCIB National Collection of Industrial Bacteria. DSM Deutsche Sammlung von Mikroorganismen. NCDO National Collection of Dairy Organisms.
Table 2. Growth conditions for all bacterial strains used.
Bacterial Strain Broth/ Days Temp °C agar Growth
Rhodococcus Species
R. coprophilus BHI broth 8 30
R. equi BHI broth 5 35
R. fascians TSA 5 30
R. marinonascens TSA 5 30
R. rhodnii TSA 6 30
R. rhodochrous TSA 6 30
R. rhodochrous TSA 6 30
R. ruber TSA 6 30
R. zopfii TSA 6 30
Rhodococcus Related Species
Actinomyces naeslundii BHI broth 6 37
Corynebacterium xerosis BHI broth 6 37
Gordona bronchialis BHI broth 6 37
Nocardia brasiliensis BHI agar 6 37
Streptomyces griseus BHI broth 6 37
Other Bacteria
Aeromonas hydrophila BHI broth 2 35 Bacillus cereus BHI broth 2 35 Bacillus subtilis BHI broth 2 35 Enterobacter aerogenes BHI broth 1 35 Enterococcus faecalis BHI broth 2 35 Escherichia coli BHI broth 1 35 Morganella morganii BHI broth 2 35 Pseudomonas aeruginosa BHI broth 2 35 Staphylococcus aureus BHI broth 2 35 Staphylococcus epidermidis BHI broth 2 35
BHI - Brain Heart Infusion. TSA - Tryptic Soy Agar
BHI agar: 5.3 g BHI agar in 100 mis distilled water. Autoclave 121 °C for 15 mins.
BHI broth: 3.8 g BHI broth in 100 mis distilled water. Autoclave 121 °C for 15 mins. Tryptic soy agar: 4 g in 100 ml distilled water . Autoclave 121°C for 15 mins. 4% Acrylamide Gel
Gel plate preparation
Both plates were washed with Jif and rinsed with dd H2O and then with 95% ethanol. The good side of small plate was covered with repel silane and the good side of the large plate with bind silane. 4mm plastic spacers were placed on the sides of the large plate and a strip of 4mm paper along the bottom. The plates were sandwiched together with gel tape.
Gel preparation
The urea was prepared while the plates were taped. 42.0 g urea was added to 36 mis of dd H2O and warmed to dissolve. To the urea the following was added:
lO ml lOx Sanger TBE 16.5 mis 40 % acrylamide 36μl TEMED
and made up to 100 mis with dd H2O. 350 μl of 10 % ammonium persulphate was added, and the acrylamide mixture drawn up into a syringe and poured slowly down one side of the tilted plate. Any bubbles were tapped out and the comb positioned. The plates were clamped, covered with Gladwrap and left overnight to polymerise.
The gel was run with loading buffer in 1 x Sanger TBE for 30 mins at 1800V, 40 mA, 50W). 8 μl of sample was added to 6 μl of loading buffer and denatured for 4 minutes at 94 °C. The wells of the gel were flushed with 1 x Sanger TBE to remove urea and 8 μl of sample was added per well. The gel was soaked in ethidium bromide/Sanger TBE (200 μl in 1 litre) but the bands were too faint to visualise and the gel was silver stained instead.
Silver staining
The gel was first fixed in 2L of 10 % glacial acetic acid for 30 minutes. After 3 washes in dd H2O it was agitated in staining solution (2 g silver nitrate and 3 ml formaldehyde in 2 L water) for 30 minutes. The gel was placed in IL of developing solution (60 g sodium carbonate in 2 L water and chilled to 10 °C. Immediately before use 3 ml of 37% formaldehyde was added and 400 μl sodium thiosulphate 10 mg/ml) for 2-3 minutes. The developing solution was then replaced with the remaining 1 L and the gel agitated for another 2-3 minutes. It was then rinsed twice in water and dried vertically over night.
PCR Assay
PCR amplification was performed in 0.5 ml tubes in a total reaction volume of 100 μl using 50 mM KCl, 10 mM Tris and 2.5 mM MgCl2 pH 8.4, 5 pmoles of each primer (0.05 μM), 2.5 Units of Taq and 200 μM of each dNTP.
Premix 1 Tube lOx PCR Buffer
(500mM KCl, 100 mM Tris, 25 mM MgCl2)10.0 μl Forward primer 1.0 μl
Reverse primer 1.0 μl dNTP's (200 μM each) 0.8 μl
Taq (2.5 units) 0.5 μl
DNA 2.0 μl dd H2O 84.7 ul
Total volume 100.0 μl
The reaction mixture was overlaid with 50 μl of nujol oil and 2 μl of DNA was added (200 ng/100 μl). The tubes were then briefly centrifuged and then they were placed in a programmable DNA thermal cycler (Perkin-Elmer Thermal Cycler 480).
The thermal profile was 94°C denaturing for 1 min, 55°C annealing for 1 min, 72°C extension for 1 min, over 30 cycles followed by a final 8 min extension step at 72°C.
Detection of PCR products
PCR products were analysed by gel electrophoresis using 2% agarose gels in TBE buffer.
10 X TBE Buffer: 0.9M Tris
0.9 M Boric acid
0.02 M EDTA pH 8.0.
Working TBE (1 X TBE) : 100 mis 10 X TBE
900 mis ddH2O
25 μl EtBr (10 mg/ml)
2% Agarose gel: 10 g agarose
50 mis 10 X TBE
450 mis ddH2O
25 μl EtBr (10 mg/ml)
Gels were run for 75 mins at 100 V in 1 x TBE buffer containing ethidium bromide to enable visualisation of the PCR products by u.v. transiUumination. Molecular weight markers were included on each gel (123 bp DNA ladder Life Technologies).
APC film development
The gel was placed on the light box gel side up and the film placed onto the gel glossy white (emulsion) side down and exposed for 20 sees. Grey side up the film was placed in developer until the bands appeared. It was washed in water, fixer and again in water.
Results
See next page for gel results. Lane 1 123 bp ladder 2 143F-467R - -. coprophilus
3 143F-467R N. brasiliensis
4 143F-1124R .fi. coprophilus
5 143F-1124R G bronchialis
6 143F-1124R C. xerosis
7 143F-1124R N. brasiliensis
8 143F-926R - ?. coprophilus
9 143F-926R S.graew.y
10 143F-926R G. bronchialis
11 143F-926R N brasiliensis
12 143F-1220R /-. coprophilus
13 123 bp ladder
14 143F-1220R S.griseus
15 143F-1220R G bronchialis
16 143F-1220R C. xerosis
17 143F-1220R N. brasiliensis
18 443F-1124R .. coprophilus
19 443F-U24R Requi
20 443F-1124R morganii
21 443F-U24R S.griseus
22 443F-1124R G. bronchialis
23 443F-1124R C. xerosis
24 443F-1124R N. brasiliensis
25 123 bp ladder
From this it was determined that no significant difference in band size could be seen and that 1124R would not be suitable for use as a primer.
DNA Extraction
Crude Extraction
10 ml cultures were centrifuged for 40 mins at 3300 g. The supernatant was removed and 300 μl extraction buffer (25 mM Tris, 10 mM EDTA, 50 mM glucose, pH 8.0. Autoclaved before use) added. The contents were transferred to a microcentrifuge tube and 20 μl lysozyme (50 mg/ml in dd H2O) was added and incubated for 5 minutes at room temperature. 12 μl 20 % SDS and 4 μl proteinase K (10 mg/ml in ddH2O) were then added and incubated at 37°C for 30 mins.
Phenol chloroform preparation
To 500 g phenol (BDH), 500 ml chloroform with 20 ml iso-amylalcohol and 0.5 g 8- hydroxyquinoline was added and left overnight at room temperature. Sufficient 0.1 M Tris HCl (pH 8.0) was added to nearly fill the container and the container shaken gently to equilibrate. The layers were allowed to separate and the top aqueous layer discarded. The mixture was washed twice more with 0.1 M Tris HCl (pH 8.0) and the pH of the supernatant checked to be between 7.5 and 8.0. The phenol/chloroform was store at 4°C under 0.1M Tris HCl (pH 8.0).
Nucleic acid extraction 300 μl of the above phenol chloroform was added to the lysed culture and gently mix end over end for 10 mins. It was then centrifuge for 15 mins at 13,000 rpm and the top aqueous layer transferred to another tube. A further 300 μl of phenol chloroform was added and mixed end over end for 10 mins. The extract was then centrifuged as before and the top aqueous phase transferred to a new tube. 300 μl of chloroform was added and mixed end over end for 10 mins. It was centrifuge as before, the top aqueous phase transferred to a new tube and 25 μl of 3M sodium acetate pH 5.2 was added with 600 μl of absolute ethanol. Tubes were then stored either overnight at -20°C or for at least 1 hour at -70°C. The tubes were centrifuged again at 13,000 rpm for 15 mins and the supernatant discarded. 600 μl of 70 % ethanol was added and centrifuged again as before. As much as possible of the supernatant was removed with a pipette and any remaining ethanol evaporated by placing the tube on a 100°C hot block until the tube was dry. The DNA was then resuspended in 20μl of dd H2O and stored at -20°C.
DNA Quantitation
To quantitate the amount of DNA present two absorbances were necessary, one at 260 nm and the other at 280 nm. 2 μl of the stock DNA solution was added to a quartz cuvette with 2 ml of ddH2O. Using ddH O as a blank the absorbances at 260 nm and at 280 nm were read.
From the A260 the concentration of DNA was determined:
1 OD unit A260 = 50 μg/ml double stranded DNA [DNA] μg/ml = A260 x 50 x 1000 (dilution)
From the A260/A280 ratio an estimate of the purity of the DNA was determined. Pure preparations of DNA have A260/A280 ratios of 1.8.
Table 3. Extraction 1. R. coprophilus.
Figure imgf000014_0001
Some of the above samples were too dilute too measure the amount of DNA present spectrophotometrically and they were run on a 1% agarose gel to check that there was DNA present. (See Figure 19) These samples were used directly in PCR and not diluted.
Table 5. Extraction 3.
Figure imgf000015_0001
Table 6. Extraction 4. Rhodococcus species
Figure imgf000015_0002
Preparation of DNA for PCR
If the stock sample of DNA was greater than 100 ng/ml(0.1 mg/ml) a working solution was prepared by dilution of the stock to 100 ng/ml. 2 μl of this working solution was added to each PCR assay to give a final concentration of 200 ng/100 μl. If the DNA was more dilute than this it was used neat in the PCR reaction, 2 μl being added. Oligonucleotide primers
Previous searches for DNA sequence data for R. coprophilus have yielded very little information. Since then however a large number of genetic sequences has been entered onto the Genbank database (http://ncbi.nlm.nih.gov/genbank) largely following an extensive study by Rainey F. A. et al 1995 on the 16S rRNA sequences for 32 strains of 26 species of the genera Rhodococcus and Nocardia.
Having collated all the 16S rRNA sequence data possible from Genbank the sequences were aligned using the DNAMAN sequence alignment package. From the alignment, areas specific to R. coprophilus could be chosen and PCR primers designed around these areas. (See appendix 2 for sequence data related to the primers used)
Eight primers were chosen, 4 selected to be specific for R. coprophilus and another four which were specific for all bacteria to be used to determine which of the R. coprophilus specific primers were working.
Table 8 showing details of 16S primers chosen
Figure imgf000016_0001
* Tm calculated by Oligo ** Tm calculated by manufacturer Life Technologies bp = base pairs
Figure 1 shows R. coprophilus 16S rRNA and associated primers.
ddH O was added to the primers to give a stock concentration of 100 nmoles/ml. A further 1/20 dilution of each primer was done to produce a working solution for PCR of 5 pmoles/μl.
From the sequence we used (Rainey F et al, 1995), the primer positions are as follows:
5' end of the 143F primer starts on base pair 143
5' end of the 419F primer starts on base pair 419
5' end of the 443F primer starts on base pair 443
5' end of the 467R complement starts on base pair 467
5' end of the 568R complement starts on base pair 568
5' end of the 1124R complement starts on base pair 1124. PCR
R. coprophilus. R. eaui and unrelated genera
Initially the first step was to test the different combinations of primers with R. coprophilus to determine that bands would be obtained. R. coprophilus was grown as stated in 'Bacteria and Cultivation' and the DNA extracted as in 'DNA extraction' extraction 1. The PCR was carried out according to 'PCR assay' above. The procedure was then repeated for all the other species outlined in Table 9, using the DNA from extraction 2. All PCR products were detected using agarose gels and visualised using ethidium bromide as outlined in 'Detection of PCR products'. The results are summarised below.
Table 9. Summary of PCR products (bp) obtained with different combinations of primers with R. coprophilus, R. equi and some commonly found bacteria.
Figure imgf000017_0001
ND = Not determined
From Table 9 it can be seen that primer 143F can distinguish between R. coprophilus and all the bacteria tested including the closely related R. equi, by the absence of a band whether it is used with or without a specific primer. From the DNA sequence alignment, for 143F to be able to distinguish between R. coprophilus and R. equi it must be able to pick up a difference of 3 bp on the 3' end.
SUBSTITUE SHEET (Rule 26) Effect of temperature.
It was thought that the two sets of primers may bind more specifically if the annealing temperature was raised from 55°C to 60°C. The PCR was repeated for these three sets of primers with all the bacterial DNA that produced a band and all the related genera. The PCR conditions were as outlined in 'PCR assay' with the exception that the annealing temperature was raised from 55°C to 60 °C
Figure imgf000018_0001
Temperature improved some of the specificity but was reduced in others.
R. coprophilus and related genera
The next set of experiments was aimed at testing more closely related species of bacteria. DNA was extracted as before from five more closely related species (extraction 3), Actinomyces naeslundii, Gordona bronchialis, Corynebacterium xerosis, Norcardia brasiliensis & Streptomyces griseus. Table 11. PCR results for Rhodococcus coprophilus and 5 related genera.
Figure imgf000019_0001
From Table 11. it can be seen that the primers are having more difficulty distinguishing between R. coprophilus and the more closely related species. It was decided to concentrate on the most likely sets of primers that would work, 143F-467R, 143F-1124R and 443F-1124R.
Sequences of the DNA at the position of the 143F primer in some closely related Rhododcoccus species.
5' 3'
R. coprophilusOSM 43357T GGGTCTAATACCGGATATGACCAT
R. equi ATCC 6939 GGGTCTAATACCGGATATGAGCJC
R. marinonascens DSM 43752T GGGTCTAATACCGGATACGACCTT R.fascians DSM 20669 GGGTCTAATACCGGATATGACCAC
If 143F is to be used it must be capable of picking up smaller differences such as those shown above for R. marinonascens and R. fascians. Both these cultures were ordered as these should be some of the most difficult to differentiate.
Primer 467R can only be used in conjunction with 143F and as 143F to date has always worked it can't be determined if 467F is working or not. Therefore another non specific forward primer was ordered, 27F, to test whether it is working.
Primer 443 F is unable to distinguish R. coprophilus from other species and therefore 143F is preferred as the forward primer. Another forward primer was ordered to replace it, 419F.
Primer 1124R can't always distinguish between R. coprophilus and other species although it often produces several bands which could be a way of distinguishing or it may be that the bands it does produce are of different lengths. All the PCR products from 1124R and 443F were run on a 4% acrylamide gel to determine if there is any difference in band size. From this it was determined that no significant difference in band size could be seen and that 1124R would not be suitable for use as a primer. A replacement primer for 1124R was ordered, 568R. Table 10. Details of new primers
Figure imgf000020_0001
New Primers (419F and 568R - Related genera/species
The new primers were diluted to give a stock concentration of 100 nmoles/ml. A further 1/20 dilution was carried out to give a working solution of 5 pmoles/μl.
PCR amplification was performed as outlined in 'PCR assay' with the following exception. The thermal profile on the Perkin-Elmer was: 94°C denaturing for 1 min, 60°C annealing for 1 min, 72°C extension for 1 min, over 30 cycles followed by a final 8 min extension step at 72°C.
Figure 2 shows PCR using new primers, 419F - 1124R and 419F - 568R.
Figure 3 shows PCR using new primers, 143F - 568R.
Large amounts of primer dimer are seen in 419F-1124R. No primer dimer is seen with 419F-568R or 143F-568R. The latter two primers show good strong bands with R. coprophilus but not with any other of the related families tested. As 419F-1124R gave bands with N. brasiliensis work was continued with the other two sets of primers(143F-568R and 419F-568R).
New Primers (143F-568R: 419F-568R) - unrelated genera
The next step was to test the two new sets of primers with the non related species.
A PCR was set up for both sets of primers using the same conditions as for the last experiment.
Figure 4 shows Primers 419F-568R with non related genera.
Figure 5 shows Primersl43F-568R with non related genera.
Both sets of the primers perform well with all non related species. The only band formed was one with B. cereus with 419F-568R, which was of a different size and easily distinguishable from the R. coprophilus band. R . coprophilus DNA was also tested in a multiplex of 143F, 419F and 568R which gave two strong bands at 425 and 149 base pairs (lane 14).The next step of the assay is to test both sets of primers with closely related Rhodococcus species to determine if there is any non specific bands.
New Primers (143F-568R: 419F-568R) - Rhodococcus species
The DNA was extracted as before (extraction 4) and a PCR set up using the same conditions as used previously. Figure 6 shows Primers 143F - 568R and 419F - 568R with some Rhodococcus species.
143F -568R showed a strong band with R.coprophilus and only a faint band with R. zopfii all other species tested showed a negative reaction. 419F-568R showed a strong band with R. coprophilus but weak bands with all others tested. It was thought that these weak bands may be removed by increasing the temperature even further to 65°C or by changing the MgCl2 concentration. Temperature was thought to be the main effector and so the experiment was repeated exactly as above except at an annealing temperature of 65°C and not 60°C.
Figure 7 shows Primers 143F - 568R and 419F - 568R with some Rhodococcus species at 60 °C.
The increase in temperature removed the non specific R. zopfii band with 143F-568R primers, however bands could still be seen with R. rhodochrous DSM 43241 and R. zopfii with primers 419F-568R. As temperature was thought to be the major factor to effect specificity, work was now only continued with primer set 143F-568R.
Optimisation of the PCR protocol - using 143F and 568R as primers
Having established a basic PCR assay that was specific for R. coprophilus, it was necessary to optimise the assay conditions to further improve the specificity of the reaction.
Temperature
An increase in temperature from 60°C to 65°C had a dramatic effect on the specificity. However it was thought possible that the temperature may be too high. Usually an annealing temperature of less than 5°C of the Tm for each primer should be used. (Tm 67°C for 143F and 71°C for 568R) A range of temperatures was tried between 60°C and 65 °C to determine the optimum, ie the sharpest and most intense R. coprophilus band and no R. zopfii band.
PCR's for R. coprophilus and R. zopfii were carried out at annealing temperatures of 61°C, 62°C, 63 °C and 64°C. Apart from the annealing temperature the conditions were identical to the previous experiments. (PCR products for 60°C and 65°C from the previous experiment were run on the gel also)
Figure 8 shows the effect of temperature on R. coprophilus and R. zopfii.
The R. coprophilus band starts decreasing in intensity at 64°C so 63 °C would be optimum, but there is possibly a faint band of R. zopfii at this temperature. To allow for changes in temperature on other machines and for the possibility of other Rhodococcus species having a stronger band the temperature is to be kept at 65°C.
MgCL concentration
lOx PCR buffer with no MgCl was prepared (500 mM KCl, 100 mM Tris) and autoclaved. 25 mM MgCl was also prepared and autoclaved. A range of MgCl concentrations was tried around the concentration already being used.
Figure 9 shows the effect of MgCl2- Wide range of concentrations. A narrower range was then tried at 2.0, 2.2, 2.4, 2.6, 2.8 & 3.0 mM
Figure 10 shows the effect of MgCl2 - Narrow range of concentrations.
It was decided that the original concentration of 2.5 mM MgCl was optimum.
Primer concentration
The primer concentration used throughout the experiments was 5 pmoles/ 100 μl. To optimise the primers a range was tried of 2, 4, 5, 6, 8 & 10 pmoles/100 μl.
Figure 11 shows the effect of primer concentration
From the above results a narrower range was tried of 2, 2,5, 3.0, 3.5, 4.0, 4.5 and 5 pmoles.
Figure 12 shows a narrower range of primers.
DNA concentration
Initially concentration of DNA is 200 ng/100 μl. A range was tried from 400 ng to 20 ng as shown below:
Figure 13 shows the effect of DNA concentration.
A lower range was then tried from 200 ng / lOOμl to 0.2 ng /100 μl
Figure 14 shows the effect of DNA concentration (lanes 9-17 only)
dNTP concentration
The usual dNTP concentration of 25mM (final cone, of 200 μM/100 μl) was further diluted 1/10 to allow a range of concentrations to be tried from 50, 100, 150, 200, 250, 300 μM.
Figure 15 shows dNTP Optimisation.
It was thought that no DNA was added to the PCR tube for the sample in lane 3. The PCR was repeated to narrow down the range and to repeat 150 μM.
Figure 16 shows fine tuning dNTP concentration.
150 μM was chosen as the optimum.
Tag polymerase concentration
Taq is currently used at 2.5 Units/100 μl and therefore a range was tried around this at, 3, 2.5, 2.0, 1.5, 1.0 and 0.5 Units.
Figure 17 shows Taq Optimisation.
2.5 Units is to be used. Final PCR Rhodococcus and related genera.
From the optimisation experiment the initial conditions were found to be optimum and the only change added was a decrease in the level of dNTP's in the final amplication mix to a 150 μM solution.
PCR amplification was performed in 0.5 ml tubes in a total reaction volume of 100 μl using 50 mM KCl, 10 mM Tris and 2.5 mM MgCl2 pH 8.4, 5 pmoles of each primer (0.05 μM), 2.5 Units of Taq and 150 μM of each dNTP.
Premix 1 Tube 1 Ox PCR Buffer
(500mM KCl, 100 mM Tris, 25 mM MgCl2)10.0 μl
Forward primer (5 pmoles/μl) 1.0 μl
Reverse primer (5 pmoles/μl) 1.0 μl dNTP's (25mM each) 0.6 μl
Taq (2.5 units) 0.5 μl
DNA (100 ng/μl) 2.0 μl dd H2O 84.7 ul
Total volume 100.0 μl
The reaction mixture was overlaid with 50 μl of nujol oil and 2 μl of DNA was added (200 ng/100 μl). The tubes were then briefly centrifuged and then they were placed in a programmable DNA thermal cycler (Perkin-Elmer Thermal Cycler 480).
The thermal profile was 94°C denaturing for 1 min, 65°C annealing for 1 min, 72°C extension for 1 min, over 30 cycles followed by a final 8 min extension step at 72°C.
All Rhodococcus and Rhodococcus related species were run including R. fascians and R. marinonascens that had not been tested before.
Discussion and Conclusions
Of all the eleven primers ordered only two reproducibly gave a R. coprophilus specific amplification, these two were 143F and 568R. All the other primer set combinations produced bands either with the related genera or with the other Rhodococcus species. All the amplification conditions have been optimized so that the resultant band is very strong.
The related genera were chosen from dendrogram family trees based on the 16S rRNA homology and similarly the Rhodococcus species were chosen in the same manner. The species chosen were ones that were either very closely related in DNA homology to R. coprophilus or selected species or genera indicative of other branches from the dendrogram. The extreme genera (little or no homology) were also tested. Obviously not all genera or species were tested, however we believe we have selectively chosen a good representation of the field and have covered the species/genera most likely to cause false positives (most closely related). An example of a genera not tested was Gordona terrae (since renamed R. terrae) however the DNA homology of this genus and R. coprophilus would indicate that there would be no reaction with the selected primers (143F and 568R). This conclusion is strengthened by the experimental observation that R. zopfii which is more closely related to R. coprophilus than R. terrae did not amplify with these primers.
Primers :
5' 3'
R. coprophilus at site of 143F: GGGTCTAATACCGGATATGACCAT R. terrae: GGGTCTAATACCGGATATGACCAA R. zopfii: GGGTCTAATACCGGATATGACCAA
R. coprophilus at site of 568R: GCAGTTGAGCTGCGGGATTTCACAC R. terrae: GCAATTGAGTTGCAGAATTTCACAG R. zopfii: GCAGTTGAGCTGCGGGTTTTCACAG
We believe that the mix of genera and species chosen, based on the published work of Rainey et al 1995 which includes most of the Rhodococcus and related genera, has included any potential species which may cause false positives.
The Rhodococcus related genera not tested are shown below with the DNA sequences at the priming sites used (143F and 568R):
Rhodococcus coprophilus # 143F GGGTCTAATACCGGATATGACCAT Rhodococcus marinonascens ** 143F GGGTCTAATACCGGATACGACCTT Rhodococcus fascians ** 143F GGGTCTAATACCGGATATGACCAC Tsukamurella paurometabolum 143F GGGTCTAATACCGGATATGACCTT Nocardia brasiliensis ** 143F GGGTCTAATACCGGATATGACCTT
Nocardia transvalensis 143F GGGTCTAATACCGGATATGACCAC Nocardia otitidis-caviarum 143F GGGTCTAATACCGGATATGACCTT Nocardia farcinica 143F GGGTCTAATACCGGATATGACCTT Nocardia calcarea 143F GGGTCTAATACCGGATATGACCTC Nocardia corynebacteroides 143F GGGTCTAATACCGGATAGGACTGC Nocardia carnea 143F GGGTCTAATACCGGATATGACCTC Nocardia asteroides 143F GGGTCTAATACCGGATATGACCTT Nocardia restrica 143F GGGTCTAATACCGGATATGAGCTC
Mycobacterium chlorophenolicum 143F GGGTCTAATACCGAATAGGACCAC Dietzia maris 143F GGGTCTAATACCGGATATGAACTC
Corynebacterium glutamicum 143F GGGTCTAATACCGAATATTCACAC Gordona sputi 143F GGGTCTAATACCGAATATTCATTT Gordona rubropertinctus 143F GGGTCTAATACCGGATATGACCTT Gordona amarae. 143F GGGTCTAATACCGGATATGACCTG
Rhodococcus coprophilus # 568R GCAGTTGAGCTGCGGGATTTCACAC Rhodococcus marinonascens ** 568R ACAGTTGAGCTGTCAGTTTTCACAA Rhodococcus fascians ** 568R GAAGTTGAGCCCCGGGTTTTCACAA Tsukamurella paurometabolum 568R GAGGTTAAGCCTCGGGTTTTCACAG Nocardia brasiliensis ** 568R GGGGTTGAGCCCCAAGTTTTCACGG Nocardia transvalensis 568R GGGGTTGAGCCCCAAGTTTTCACGA Nocardia otitidis-caviarum 568R GGGGTTGAGCCCCAAGTTTTCACGG Nocardia farcinica 568R GGGGTTGAGCCCCAAGTTTTCACGG Nocardia calcarea 568R GCAGTTGAGCTGCTGGTTTTCACAA Nocardia corynebacteroides 568R ACAGTTGAGCTGCTGGTTTTCACAG Nocardia carnea 568R GGGGTTGAGCCCCGAGTTTTCACGA Nocardia asteroides 568R GGGGTTGAGCCCCAAGTTTTCACGA Nocardia restrica 568R GGGGTTGAGCCCCAAGTTTTCACGG
Mycobacterium chlorophenolicum 568R ACAGTTAAGCTGTGAGTTTTCACGA
Dietzia maris 568R CCGGTTAAGCCGAGGGATTTCACAG
Corynebacterium glutamicum 568R GAAGTTAAGCCCNGGGATTTCAAAG
Gordona sputi 568R GCAGTTAAGCTGCAGAATTTCACAG
Gordona rubropertinctus 568R ACAATTGAGTTGCAGAATTTCACAG
Gordona amarae 568R ACAATTGAGTTGCAGAATTTCACAG
** Denotes genera or Rhodococcus species that have been tested with this method and found not to give false positives. They have been included as markers to allow comparison of sequence differences which can be tolerated in this method.
# Denotes R. coprophilus and the DNA sequence for which this method has been designed at the primer sites.
All tested species with closely related DNA sequences were found not to react. From the above list and Appendix 2 most bacteria not tested have more mismatches in the primer regions than those tested. Therefore these species are highly unlikely to cause false positives. All of the genera and Rhodococcus species not tested were positioned closely in the DNA dendrogram to other bacteria that tested negative. For this reason we believe that false positives will not occur. For example Tsukamurella paurometabolum has a close DNA sequence to R. coprophilus at the 143F primer site. However this sequnce is very similar to N. brasiliensis which was found not to give a product.
Of the Nocardia, N. transvaliensis, only has one mismatch on the DNA sequence at the 143F primer site, however within the DNA sequence in the 568R primer region many mismatches occur and for this reason we do not believe amplification is possible. Other bacteria such as N. calcarea and N. corynebacteroides have few mismatches in the 568R primer region but more in the 143F region. Of the remainding genera Gordona rubropertinctus is the most similar to R. coprophilus. However all of the genera have more mismatches than R. zopfii and therefore highly unlikely to amplify.
The following list contains those genera and Rhodococcus species which have not been tested but potentially need to be investigated if any problems occur:
R. opacus DSM 43206T R. erythropolis ATCC 4277T
R. luteus DSM 43673 R. globerulus DSM 4954T
G. terrae DSM 43249 N. calcarea DSM 43188T
N. transvalensis DSM 43405T N. corynebacteroides DSM 2015 IT
T. paurometabolum DSM 20162T R. chubuensis DSM 44019T
G. rubropertinctus DSM 43197T From the above it can be seen that a reliable assay has been developed producing strong bands with R. coprophilus. From the DNA sequences obtained this assay is highly unlikely to react with any other bacteria. No amplification products have been found with any other species tested to date other than R. coprophilus.
Figure 18 shows 143F-568R with Rhodococcus and Rhodococcus related genera
Figure 19 shows DNA extraction 2 run on a 1% agarose gel
Figure 20 shows phylogenetic dendrogram based on the comparison of 16S rRNA sequences of Rhodococcus and Rhodococcus related genera.
Figures 21 shows the results of the PCRs with Rhodococcus coprophilus DNA and different sets of primers.
B. Development of a PCR method for the detection of Listeria monocytogenes
Experimental
Bacterial Strains & Cultivation
The bacterial strains included in this study are listed in Table 1.
Table 1
Bacterial Strain Source
Listeria Species
Listeria monocytogenes ATCC 19111 Listeria innocua ATCC 33090 Listeria ivanovii CDC 797 Listeria seeligeri ATCC 35967
Listeria Related Species
Bacillus cereus ATCC 10702, NCTC 8035, NCIB 8122 Bacillus subtilis ATCC 6051, NCTC 3610, NCIB 3610 Staphylococcus aureus ATCC 25923
Other Bacteria
Aeromonas hydrophila ATCC 7966, NCTC 8049, NCIB 9240 Campylobacter jejuni ATCC 33560 Enterobacter aerogenes ATCC 13048, NCTC 10006 Enterococcus faecalis ATCC 19433, NCTC 775, NCDO 581 Escherichia coli ATCC 25922 Morganella morganii ATCC 25830, NCTC 235 Rhodococcus coprohilus ATCC 29080, NCTC 10994, DSM 43347T Pseudomonas aeruginosa ATCC 25668, NCTC 10662 Salmonella menston CDC 383 Shigella flexneri CDC 972 Shigella sonnei CDC 86 Staphylococcus epidermidis ATCC 12228 Yersinia enterolitica ATCC 9610
ATCC American Type Culture Collection. NCTC National Collection of Type Cultures. NCIB National Collection of Industrial Bacteria. DSM Deutsche Sammlung von Mikroorganismen. NCDO National Collection of Dairy Organisms. CDC Communicable Disease Centre New Zealand. Table 2. Growth conditions for all bacterial strains used.
Bacterial Strain Broth/ Days Temp agar Growth °C
Listeria Species
Listeria monocytogenes BHI broth 1 35
Listeria innocua BHI broth 1 35
Listeria ivanovii BHI broth 1 35
Listeria seeligeri BHI broth 1 35
Listeria Related Species
Bacillus cereus BHI broth 2 35
Bacillus subtilis BHI broth 2 35
Staphylococcus aureus BHI broth 2 35
Other Bacteria
Aeromonas hydrophila BHI broth 2 35
Campylobacter jejuni BHI broth 1 35
Enterobacter aerogenes BHI broth 1 35
Enterococcus faecalis BHI broth 2 35
Escherichia coli BHI broth 1 35
Morganella morganii BHI broth 2 35
Rhodococcus coprophilus BHI broth 8 30
Pseudomonas aeruginosa BHI broth 2 35
Salmonella menston BHI broth 1 35
Shigella flexneri BHI broth 1 35
Shigella sonnei BHI broth 1 35
Staphylococcus epidermidis BHI broth 2 35
Yersinia enterolitica BHI broth 1 35
BHI - Brain Heart Infusion. TSA ■ - Tryptic Soy Agar
BHI agar: 5.3 g BHI agar in 100 mis distilled water. Autoclave 121 °C for 15 mins.
BHI broth: 3.8 g BHI broth in 100 mis distilled water. Autoclave 121 °C for 15 mins.
Tryptic soy agar: 4 g in 100 ml distilled water . Autoclave 121°C for 15 mins.
DNA Extraction
Crude Extraction
10 ml cultures were centrifuged for 40 mins at 3300 g. The supernatant was removed and 300 μl extraction buffer (25 mM Tris, 10 mM EDTA, 50 mM glucose, pH 8.0. Autoclaved before use) added. The contents were transferred to a microcentrifuge tube and 20 μl lysozyme (50 mg/ml in dd H O) was added and incubated for 5 minutes at room temperature. 12 μl 20 % SDS and 4 μl proteinase K (10 mg/ml in ddH2O) were then added and incubated at 37°C for "30 mins.
Phenol chloroform preparation
To 500 g phenol (BDH), 500 ml chloroform with 20 ml iso-amylalcohol and 0.5 g 8- hydroxyquinoline was added and left overnight at room temperature. Sufficient 0.1 M Tris HCl (pH 8.0) was added to nearly fill the container and the container shaken gently to equilibrate. The layers were allowed to separate and the top aqueous layer discarded. The mixture was washed twice more with 0.1 M Tris HCl (pH 8.0) and the pH of the supernatant checked to be between 7.5 and 8.0. The phenol/chloroform was store at 4°C under 0.1M Tris HCl (pH 8.0).
Nucleic acid extraction
300 μl of the above phenol chloroform was added to the lysed culture and gently mix end over end for 10 mins. It was then centrifuge for 15 mins at 13,000 rpm and the top aqueous layer transferred to another tube. A further 300 μl of phenol chloroform was added and mixed end over end for 10 mins. The extract was then centrifuged as before and the top aqueous phase transferred to a new tube. 300 μl of chloroform was added and mixed end over end for 10 mins. It was centrifuge as before, the top aqueous phase transferred to a new tube and 25 μl of 3M sodium acetate pH 5.2 was added with 600 μl of absolute ethanol. Tubes were then stored either overnight at -20°C or for at least 1 hour at -70°C. The tubes were centrifuged again at 13,000 rpm for 15 mins and the supernatant discarded. 600 μl of 70 % ethanol was added and centrifuged again as before. As much as possible of the supernatant was removed with a pipette and any remaining ethanol evaporated by placing the tube on a 100°C hot block until the tube was dry. The DNA was then resuspended in 20μl of dd H2O and stored at -20°C.
DNA Quantitation
To quantitate the amount of DNA present two absorbances were necessary, one at 260 nm and the other at 280 nm. 2 μl of the stock DNA solution was added to a quartz cuvette with 2 ml of ddH O. Using ddH2O as a blank the absorbances at 260 nm and at 280 nm were read.
From the A260 the concentration of DNA was determined:
1 OD unit A260 = 50 μg/ml double stranded DNA [DNA] μg/ml = A260 x 50 x 1000 (dilution)
From the A260/A280 ratio an estimate of the purity of the DNA was determined. Pure preparations of DNA have A260/A280 ratios of 1.8.
Table 3. Extraction 1. L. monocytogenes
Figure imgf000029_0001
Table 4. Extraction 2.
Figure imgf000030_0001
Preparation of DNA for PCR
If the stock sample of DNA was greater than 100 ng/ml(0.1 mg/ml) a working solution was prepared by dilution of the stock to 100 ng/ml. 2 μl of this working solution was added to each PCR assay to give a final concentration of 200 ng/100 μl. If the DNA was more dilute than this it was used neat in the PCR reaction, 2 μl being added.
Oligonucleotide primers
Two pairs of primers were needed for the PCR, one pair to be specific for L. monocytogenes the other pair specific for all Listeria species.
Listeria monocytogenes specific primers For the L. monocytogenes specific primers the listeriolysin O gene was chosen as it is only found in haemolytic bacteria and would narrow down the amount of organisms that the primers would cross react with. The DNA sequence for the few Listeriolysin sequences that could be found on the Genbank database (http://ncbi.nlm.nih.gov/genbank) were aligned using the GCG software package and regions specific for L. monocytogenes were chosen for the priming sequences. Four primers were chosen that were thought to be specific for L. monocytogenes. Two other primer sequences, LF and LR that have already been published (Bansal 1996) were also selected. Details of each primer are given in Table 6.
Table 6 showing the details of the listeriolysin O primers chosen
Figure imgf000031_0001
bp = base pairs
Figure 22 shows L.monocytogenes listeriolysin O gene and associated specific primers.
Most favoured primers shown in bold giving a 706 bp product.
The primer positions are as follows: the 5 ' end of 31 OF starts on base pair 310, the 5' end of 715F starts on base pair 715, the 5 ' end of 1016R complement starts on base pair 1016, the 5 ' end of 1183R complement starts on base pair 1183.
Listeria specific primers
For the Listeria specific primers the 16S rRNA was compared from a large number of bacteria but there were very few regions that were specific to Listeria. Having collated all the 23 S rRNA sequence data possible from Genbank the sequences were aligned using the GCG sequence alignment package. From the alignment, areas specific to Listeria could be chosen and PCR primers designed around these areas.
Table 7 showing details of 23 S rRNA primers chosen
Figure imgf000031_0002
Figure imgf000032_0001
* Tm calculated by manufacturer Life Technologies bp = base pairs
Figure 23 shows Listeria 23 S rRNA gene and associated Listeria specific primers.
Most favoured primers shown in bold giving a 241 bp product
The primer positions are as follows:
the 5 ' end of L318F starts on base pair 318, the 5' end of L1541F starts on base pair 1541, the 5' end of L1993F complement starts on base pair 1993, the 5' end of L559R complement starts on base pair 559, the 5' end of L2038R complement starts on base pair 2038, the 5' end of L2534R complement starts on base pair 2534.
All primers were diluted with ddH2O to give a stock concentration of 100 nmoles/ml. A further 1/20 dilution of each primer was done to produce a working solution for PCR of 5 pmoles/μl.
PCR Assay
PCR amplification was performed in 0.5 ml tubes in a total reaction volume of 100 μl using 50 mM KCl, 10 mM Tris and 2.5 mM MgCl2 pH 8.4, 5 pmoles of each primer (0.05 μM), 2.5 Units of Taq and 200 μM of each dNTP.
Premix l Tube lOx PCR Buffer (500mM KCl, 100 mM Tris, 25 mM MgCl2) 10.0 μl
Forward primer 1.0 μl
Reverse primer 1.0 μl dNTP's (200 μM each) 0.8 μl
Taq (2.5 units) 0.5 μl
DNA 2.0 μl dd H2O 84.7 ul
Total volume 100.0 μl
The reaction mixture was overlaid with 50 μl of nujol oil and 2 μl of DNA was added (200 ng/100 μl). The tubes were then briefly centrifuged and then they were placed in a programmable DNA thermal cycler (Hybaid Omnigene).
The thermal profile was 95°C denaturing for 1 min, 55°C annealing for 1 min, 72°C extension for 1 min, over 30 cycles followed by a final 8 min extension step at 72°C.
Detection of PCR products
SUBSTITUE SHEET (Rule 26) PCR products were analysed by gel electrophoresis using 2% agarose gels in TBE buffer.
10 X TBE Buffer: 0.9M Tris
0.9 M Boric acid
0.02 M EDTA pH 8.0.
Working TBE (1 X TBE) 100 mis 10 X TBE
900 mis ddH2O
25 μl EtBr (10 mg/ml)
2% Agarose gel: 10 g agarose
50 mis 10 X TBE
450 mis ddH2O
25 μl EtBr (10 mg/ml)
Gels were run for 75 mins at 100 V in 1 x TBE buffer containing ethidium bromide to enable visualisation of the PCR products by u.v. transiUumination. Molecular weight markers were included on each gel (123 bp DNA ladder Life Technologies).
PCR - All L. monocytogenes specific primers against L. monocytogenes
Initially the first step was to test the nine different combinations of L. monocytogenes specific primers against L. monocytogenes to determine that they all give positive results. L. monocytogenes was grown as stated in 'Bacteria and Cultivation' and the DNA extracted as in 'DNA extraction' extraction 1. The PCR was carried out according to 'PCR assay' above.
Figure 24 shows Listeria monocytogenes with all L.monocytogenes specific primers.
Figure imgf000033_0002
Figure imgf000033_0001
All primer combinations gave bands with L. monocytogenes and all gave the theoretical size of product. The primer pairs that gave the strongest bands were those with 31 OF. 31 OF and 1016R were chosen to be run as a positive control for later gels. LF and LR gave a fainter band than the others but this could be due to them not being used at the optimum conditions specified by Bansal (1996) ie 1.5 mM MgC12 at 51°C annealing temperature.
The procedure was then repeated with E. coli, B. subtilis, B. cereus, S. aureus, E. faecalis and A. hydrophila to determine the specificity of the primers. B. subtilis is quite closely related to L. monocytogenes and it was thought that it may react. The DNA used was prepared from extraction 2. For all photographs of the gels see Figures 49-52. No product was formed with any of the primer pairs tested against any of the bacteria stated above.
PCR - All L.monocytogenes specific primers against some food samples
In an attempt to narrow down the number of primers used two food samples were tested. These two food samples had been previously tested in the PHL lab and one was found to be positive for Listeria monocytogenes (sample 382) and the other negative (sample 385). These two samples were tested with all nine different sets of primers. The results are shown below:
Figure 25 shows Positive and negative food samples with all L. monocytogenes specific primers.
Figure imgf000034_0001
Figure imgf000034_0002
The above experiment was repeated with two other food samples 300 and 307, both positive for L. monocytogenes.
Figure 26 shows two positive food samples with all L. monocytogenes specific primer pairs.
Figure imgf000034_0003
Figure imgf000034_0004
SUBSTITUE SHEET (Rule 26)
Figure imgf000035_0001
Figure imgf000035_0002
From the above two experiments it was found that all the L. monocytogenes specific primers chosen were working, forming PCR products with the three positive food samples and no PCR products with the negative food sample. Any of these pairs could be used at this stage but it was decided to narrow the number of primer pairs down to the four sharpest and most intense bands. The primer pairs chosen were:
715F & 1016R 301 bp. See lane 5, 14 Figure 48 715R & 1183R 468 bp. See lane 6, 15 Figure 48 310F & 1016R 706 bp. See lane 8, 17 Figure 48 310F & 1183R 873 bp. See lane 9, 18 Figure 48
PCR - Other Listeria species with L. monocytogenes specific primers
Three other Listeria species were tested against the four primer pairs, L. ivanovi, L. innocua and L. seeligeri.
Figure 27 shows Listeria species against the four different primer pairs.
Figure imgf000035_0003
Figure imgf000035_0004
No PCR product was formed with any of the three Listeria species tested with any of the four primer pairs.
PCR - Other food related bacteria with L. monocytogenes specific primers
Five food related bacteria and two L. monocytogenes positive samples were tested with each of the four primer pairs:
Figure 28 shows Shigella flexneri, Shigella sonnei and Salmonella menston with L. monocytogenes specific primers.
Figure imgf000035_0005
Figure imgf000035_0006
SUBSTITUE SHEET (Rule 26)
Figure imgf000036_0001
Figure imgf000036_0002
Figure 29 shows two L. monocytogenes positive food samples (298 & 291), Yersinia enterolitica, Campylobacter jejuni with L. monocytogenes specific primers
Figure imgf000036_0003
Figure imgf000036_0004
It was found that the positive L. monocytogenes samples 298 and 297 gave either faint bands or in two cases (lanes 2 & 3) gave no visible band at all. This was thought to be due to either inhibition by a contaminant in the food sample which may be eliminated by adding BSA to the premix, or a too dilute sample of DNA.
All species results with both sets of primers and final PCR procedure are summarised on page 42.
PCR - Effect of BSA in the premix
The two food samples from the previous experiment were retested using BSA in the PCR premix. A 2 mg/ml solution of BSA was prepared in water. 10 μl of this solution was added to each tube to give a final concentration of 0.2 mg/ml The concentration of DNA was also tested by adding double the usual volume with no BSA using 31 OF & 1016R as primers. (Lanes 6 & 12). A control of no BSA and a normal lx concentration of DNA was run using 31 OF & 1016R as primers. (Lanes 7 & 13)
Figure 30 shows Effect of BSA and DNA concetration on PCR product in food samples
Figure imgf000036_0005
SUBSTITUE SHEET (Rule 26)
Figure imgf000037_0001
Figure imgf000037_0002
It was found that all PCR products were more intense and sharper with the addition of BSA. BSA at 0.2 mg/ml is to be added to the premix in the future. For one sample the increase in DNA had no effect but the other there was a marked increase in the intensity of the band.
PCR - Listeria specific primers
The first step was to test the three sets of Listeria specificprimer pairs aginst the four Listeria species and two closely related bacteria, B. subtilis and S aureus.
Figure 31 shows Listeria specific primers against four Listeria species and two closely related bacteria.
Figure imgf000037_0003
Figure imgf000037_0004
The first primer pair L318F & L559R gave bands with all the Listeria species (lanes 2, 5, 8 and 11) and no bands with the other two related species B. subtilis(lane 14) and S. αureus(\ane 17). The second primer pair L 154 IF & L2038R gave a false positive with B. subtilis(lane 15) but not with S. αureus(\ane 18). The final pair, L1993F & L2534R gave false positives with both related bacteria(lanes 16 & 19). It was thought that an increase in the annealing temperature from 55°C to 62°C may improve the specificity.
Effect of temperature
It is thought that 5°C below the Tm of the primer is an optimum annealing temperature. The lowest Tm for the Listeria specific primers is 67°C (see Table 7)and it was therefore decided that the annealing temperature could be raised to 62°C The previous experiment was repeated as before but with an annealing temperature of 62°C.
SUBSTITUE SHEET (Rule 26) Figure 32 shows the effect of temperature on the specificity of the Listeria specific primers
Figure imgf000038_0001
Figure imgf000038_0002
It was found that increasing the annealing temperature to 62°C from 55°C eliminated the PCR products with S. aureus but two bands were still produced with B. subtilis although the L 154 IF & L2038R band was fainter.
Effect of Magnesium Chloride
One pair of Listeria specific primers (L318F & L559R) picks only the Listeria strains and could be used in the PCR assay. L 154 IF & L2038R could potentially be improved with a change in the MgCl2 concentration. No further work was to be carried out on L1993F & L2038R. A PCR was set up using lOx PCR buffer with no MgCl2 A 25 mM solution of Mg Cl2 was prepared and a range of concentrations set up between 0.5 mM and 5.0 mM. Two organisms were tested L. monocytogenes and B subtilis, a concentration was looked for that would produce a band with L. monocytogenes but not with B. subtilis. The PCR was run at an annealing temperature of 62°C.
Figure 33 shows the effect of MgCl2 on L1541F & L2038R using L.monocytogenes and B. ubtilis.
Figure imgf000038_0003
Figure imgf000038_0004
It was found that 1.5 mM MgCl2 gave a faint band with L.monocytogenes (lane 4) the minimum MgCl2 would be 2.0 mM. At this concentration there was no band with B. subtilis. Primer pair L1541F & L2038R could be used for Listeria detection if used at
SUBSTITUE SHEET (Rule 26) 2.0 mM MgCl2 concentration as a back up for L318F & L559R.
The B. subtilis 23 S rRNA was retrieved from the Genbank database and the 3 sets of primers compared to it using Oligo.lt was found that:
- L318F & L559R had no matches and would not bind the B. subtilis DNA and therefore not produce any amplification prdoducts.
- L 154 IF had 12 matches on the 3' end but a lot of mismatches on the 5 'end. L2038R had 24 of its 25 bp matching the B. subtilis DNA. This would indicate that they are quite likely to form a PCR product.
- L1993F had 1 mismatch which was not near the 3' end and L2534R had 1 mismatch near the 3; end indicating that these as a pair would almost certainly bind to B. subtilis and produce amplification products.
The theoretical findings using Oligo matched exactly with what was found experimentally, indicating that Oligo can be a very useful tool for the initial selection of primer sequences.
PCR - Multiplex
Two primer pairs have now been established individually, one specific for L. monocytogenes (31 OF & 1016R), the other for Listeria (L318F & L559R) The next step is to determine if the two primer pairs can be used together in the same PCR reaction and can distinguish between L. monocytogenes, other Listeria species and other bacteria. PCR was carried out accordong to 'PCR Assay' except that BSA was used at 0.2 mg/ml per tube and the annealing temperature was at 62°C.
Figure 34 shows multiplex (310F & 1016R, L318F & L559R)
Figure imgf000039_0002
Figure imgf000039_0001
It was found that the primer pairs worked as expected. They showed the first six samples to be L. monocytogenes positive. The seventh sample (lane 8) was a negative food sample and gave no bands on the gel. The next three were Listeria species and only the expected Listeria band formed.. The last seven organisms were not related and no PCR product was obtained. In lane 5 the bands were quite weak. The gel was rerun to see if it was a loading problem.The gel was run as before but at 70V.
SUBSTITUE SHEET (Rule 26) The gel still showed faint bands therefore they are not due to under loading.
Optimisation of MgCI2 for the multiplex
The next step was to determine the optimum concentration of MgCl2 for the multiplex. A range of MgCl2 concentrations was tried between 0.5 and 4.0 mM for primers 310F, 1016R, L318F and L559R (lanes 2-9) Other multiplex combinations were also tried (lanes 10-12):
- 31 OF, 1183R, L318F and L559R producing bands at 873 and 241
- 310F, 1016R, L1541F and L2038R producing bands at 706 and 497
- 310F, 1183R, , L1541F and L2038R producing bands at 873 and 497
Figure 35 shows optimisation of MgCl2 (lanes 2-9). Different multiplexes (lanes 10-12)
Figure imgf000040_0002
Figure imgf000040_0001
From the gel it can be seen that there is no PCR product formed at 0.5 and 1.0 mM MgCl2. The concentration is to be kept at 2.5 mM MgCl2. It was found that two of the other primer combinations worked lane 10 (310F, 1183R, L318F and L559R) and lanel l (310F, 1016R, L1541F and L2038R).Lane 12 (310F, 1183R, , L1541F and L2038R) gave a faint L.monocytogenes band and therefore may not be as good as the other three combinations.
PCR - To test the current DNA extraction procedure.
To be able to directly compare the current PCR procedure with the new one the extraction methods used must be the same. It was therefore decided to run a pure culture of L. monocytogenes DNA having been extracted using the current DNA extraction procedure. The PCR was then run according to 'PCR assay' with added BSA and an annealing temperature of 62°C. Four different primer combinations were tested (lanes 2-5). When primer pair L1541F & L2038R were used the MgCl2 concentration had to be 2.0 mM.
Figure 36 shows L. monocytogenes DNA extracted using the current procedure and tested using the different mulitplex systems.
Figure imgf000040_0003
Figure imgf000041_0001
Even when using the current extraction procedure, which doesn't give such a pure preparation of DNA, only two bands occur. No non specific bands are present. All combinations of the primers shown could be used. However the one in lane 2 is favoured as it gives the strongest bands.When the L.monocytogenes specific primers are used with L 154 IF and L2038R (lanes 4 and 5) they give a weaker band than with the other combinations. This makes them less suitable for use than the primer combination in lane 2.
PCR - Comparing known primers and primers according to the present invention
Viable and non viable L. monocytogenes cells from 3 samples had been stored frozen. The samples were defrosted and treated in three different ways (according to appendix 4 section 7.5, 7.6 and 7.7) before running the new PCR:
Viable cells: resuspended and diluted in water (7.5) denatured (7.6) centrifuged (7.7) - sample PCR'd(A)
Non viable: centrifuged (7.7)- supernatant PCR'd (C ) resuspended, denatured,(7.6) centrifuged (7.7)- supernatant PCR'd (B)
Figure 37 shows a procedure using primers 31 OF and 1016R.
Figure imgf000041_0003
Figure imgf000041_0002
A number of conclusions can be drawn from this experiment:
1. DNA can be extracted from frozen cells and still give a reliable positive result.
2. Heat denatured DNA is able to be frozen and reused with no adverse effect on the results, i.e.the expected bands still amplify and there are no non specific bands.
3. There is no need to redenature DNA, it is sufficient to recentrifuge the cells and use the supernatant.
The results from the new primers were then compared to the results from the known primers using a PCR procedure.
Figure 38 shows the results of a PCR using known primers
Figure imgf000042_0001
The known Listeria primers gives weak amplification products when the sample is at
10'9(lane 5 figure 38) however the new procedure gives strong bands at 10'9. The new primers appear more sensitive.
PCR - Comparison of new and current PCR procedures with food samples
Four food samples were run using the new procedure and compared to the results obtained using the current method. In Figure 39 the upper band (241 bp) is the Listeria band and the lower (706 bp) is the L. monocytogenes. In Figure 40 the upper band in sample 1 is the L. monocytogenes band and the lower one the Listeria band.
Figure 39 shows a method using new primers.
Figure 40 shows a method using known primers.
Figure imgf000042_0002
It is clear from the above results that the known primers give a lot of non specific amplification products that can cause confusion in reading the result. The new primers according to the present invention amplify more specifically and only gives two bands with L. monocytogenes.
It was thought that the non specific bands could be due to the extraction procedure used for the current method which only crudely purifies the DNA. To determine if this was so a further 34 food samples were tested having had the DNA extracted using the current procedure. The following results therefore are from samples that have been extracted using the current DNA extraction procedure and the only variation in the results is caused by the difference in the PCR procedures.
Figure 41 shows food samples using the new primers.
Figure 42 shows food samples using the known primers.
Figure imgf000043_0003
Figure imgf000043_0002
From the above threee of the samples (1, 2, and 3) were found to be Listeria positive but not L. monocytogenes positive. A much clearer result is obtained using the primers according to the present invention.
Figure 43 shows food samples using the primers according to the invention.
Figure 44 shows food samples using known primers.
Figure imgf000043_0005
Figure imgf000043_0004
PCR -Comparison of more food samples and some unrelated bacteria
Figure 45 shows food samples using new primers.
L C B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 C L Figure 46 shows food samples using known primers.
Figure imgf000043_0001
Figure imgf000043_0006
Figure imgf000043_0007
Figure imgf000044_0001
Figure imgf000044_0002
One sample showed Listeria positive but L. monocytogenes negative (sample 11). This result is a lot clearer using the new primers than it is using known primers.
SUMMARY
Table 8. Summary of all the pure cultures tested with the two sets of primers.
Figure imgf000044_0003
NT = Not tested. PCR procedure according to the present invention:
The final PCR procedure for the detection of Listeria monocytogenes is outlined below. PCR amplification was performed in 0.5 ml tubes in a total reaction volume of 100 μl (This can be scaled down to 20 μl to economise on materials.) using 50 mM KCl, 10 mM Tris and 2.5 mM MgCl2 pH 8.4, 5 pmoles of each primer (0.05 μM), 2.5 Units of Taq and 200 μM of each dNTP. It has also been determined that the addition of BSA at a final concentration of 0.2 mg/ml helps to prevent inhibiton of amplification by any contaminants in the samples. The annealing temperature has also been raised from 55°C to 62°C to increase the specificty of the assay.
Premix 1 Tube
lOx PCR Buffer(500mM KCl, 100 mM Tris, 25 mM MgCl2) 10.0 μl
BSA (2 mg/ml) 10.0 μl
Forward primer 31 OF 1.0 μl
Reverse primer 1016R 1.0 μl
Forward primer L318F 1.0 μl
Reverse primer L559R 1.0 μl dNTP's (200 μM each) 0.8 μl
Taq (2.5 units) 0.5 μl
DNA 2.0 μl dd H2O 72.7 ul
Total volume 100.0 μl
The reaction mixture was overlaid with 50 μl of nujol oil and 2 μl of DNA was added (200 ng/100 μl). The tubes were then briefly centrifuged and then they were placed in a programmable DNA thermal cycler (Hybaid Omnigene).
The thermal profile was 95 °C denaturing for 1 min, 62°C annealing for 1 min, 72°C extension for 1 min, over 30 cycles followed by a final 8 min extension step at 72°C.
Discussion and Conclusions
Listeria
When the primer sequences for the two Listeria specific primers, known previously, were checked against the 16S rRNA DNA alignment, primer Ul was found (region 528 -545) not to be Listeria specific but a universal primer and will bind to any bacteria. UII, the second primer (region 1566- 1587) was found to be Listeria specific on the last base pair on the 3' end with a few other mismatches throughout the sequence. The 3' end of a primer is the most important region for specificity to occur. The significance of only one mismatch on the 3' end is, the assay may have great difficulty in distinguishing between Listeria and other species particularly at the low annealing temperature used (49°C) and the higher magnesium concentration used (3 mM) both of which would promote non specific binding. This may well account for all the extra bands that have been formed during this current PHL PCR. PCR method according to the present invention using new primers: When all the 16S rRNA sequences were taken from Genbank and aligned using the GCG package very few regions common to all Listeria species, but not found in any other species were found. This is why the 23 S rRNA DNA was checked for possible Listeria specific sites. A number of Listeria specific sites were found on the 23 S rRNA which have several mismatches on the 3' end with other species and were therefore highly likely to bind specifically to Listeria. This has been confirmed with the work carried out to date
Of the three Listeria specfic primer pairs that were chosen one pair gave better amplification than the others (L318F and L559R). This primer pair gave a 241 bp product with all Listeria species tested but not with any other bacteria tested. A second primer pair (L 154 IF & L2038R) could be used if for any reason the first cannot but only if the MgCl2 concentration is at 2.0 mM
Listeria monocytogenes
PCR method according to the present invention using new primers: The listeriolysin gene which codes for the protein causing haemolysis was chosen as the region of DNA specific for the L. monocytogenes species DNA sequences were taken from Genbank for all lysin genes. We obtained sequences from 5 L. monocytogenes species, L. ivanovii and L. seeligeri and using the GCG package aligned these to examine for potential primer sites. We chose four sites and in addition included the two new sites used by Dr Bansal, LF and LR (Bansal 1996 in press). All sites (9 combinations) amplified with no cross reactivity occuring with other Listeria species. However we chose one combination based on the strength and length of the product from primers 31 OF and 1016R.
Of the nine primer pairs specific for L. monocytogenes all of them gave bands against L. monocytogenes and didn't cross react with any of the three other Listeria cultures or the closely related species tested. The primer pair that was selected (31 OF and 1016R) gave a 706 bp amplification product.
We were concerned that very few DNA sequences had been found for the lysin gene. We downloaded all the Listeriolysin sequences for all the L. monocytogenes strains listed on Genbank, 11 L. monocytogenes, 2 Listeria species 9 other non Listeria species:
Aeromonas hydrophila Streptococcus pneumoniae
Escherichia coli Streptococcus pyogenes
Proteus vulgaris Vibrio cholerae
Streptococcus canis Vibrio par ahaemolyticus Streptococcus equisimilis
The DNA was checked against the new primers (31 OF and 1016R) using the DNA program Oligo. All the L. monocytogenes species showed complete binding of both primers and all produced a 706 base pair fragment. All the other species and genera tested showed no or only an extremely small degree of potential primer binding. Theoretically the chosen primers should not bind with this DNA and certainly will not cause ambiguity of the results.
When the L. monocytogenes primers (LMI and LMII) that are currently used were checked against the DNA GCG alignment package they were found to be in regions that had degrees of specificity for L. monocytogenes. The new L.monocytogenes primers 31 OF and 1016R are in the same area as LMI and LMII but are upstream. The 31 OF primer is three bases and the 1016R primer is twelve bases upstream. Both these differences incorporate more mismatches at the 3' ends and along the whole template. In addition the primers are 25 bases not 18 bases in length. The effects of both are to increase specificity of binding to the L. monocytogenes species and obviously reducing the non specific binding observed.
The melting temperatures (TMs) of our new primers and known, published primers are shown in the following Table 9.
TABLE 9
Primer Life Technologies formula Oligo method
310F 71 63
LMI 63 47
1016R 67 54
LMI I 64 52
715F 64 51
LF 61 47
1183R 65 52 primer 2(Fluit) 63 41
This shows that our new primers have different TMs to known published primers.
A known DNA method based on earlier work of Dr Bansal used the Listeria specific primers of Ul and UII combined with the L. monocytogenes primers LMI and LMII. This combination of primers has been found to cause many non specific bands. The Listeria primers Ul and UII (UII is also known as LII in Bansal 1996) have been combined with L. monocytogenes primers LF and LR. These latter two primers are used at a lower annealing temperature of 51°C. The LR primer has no L. monocytogenes sites on the last three bases at the 3' end and the LF primer has only one specific site on the 3' end. The LF primer has therefore fewer specific sites on this 3' end than our corresponding primer and is potentially more likely to mistype than the primers according to the present invention.
The primers according to the present invention have been designed to maximise the nucleotide differences between all the existing Listeria sequences. Preexisting primers do not. The present primers are superior and significantly different to any known primers.
Of the Listeria specific primers Ul and UII only UII is Listeria specific placing all the specifitcity on one primer and not two which is preferable and which we have achieved.
When testing some food samples it was noted that some of the bands were very faint when there should have been a strong positive result. This was thought to be due to inhibiton by a contaminant in the food sample. It was found that the PCR products were more intense and sharper with the addition of BSA in the premix. BSA should now be added at 0.2 mg/ml to the premix to decrease the inhibtion by food samples. The two primer pairs have been tested in a multiplex system against forty food samples. Both DNA methods have typed these samples similarly however the majority of the current PHL DNA results were difficult to interpret with many non specific amplification bands being formed. These same samples when run in the new DNA method produced either the expected one or two bands or nothing in non Listeria samples. The absence of the non specific bands greatly increase the ease of reading the results and makes it less likely for errors to occur. It was thought that some of the non specific bands in the current PHL assay could be due to the DNA extraction which only crudely purifies the DNA. However the forty food samples tested with the new primers were extracted using the crude method and therefore the improvement seen is due solely to the new PCR method and not the extraction procedure.
The new PCR has been tested against three closely related bacteria and a number of other unrelated organisms. The extreme genera (little or no homology) were tested particularly if they were food related pathogens that may be present in the types of samples for which the PCR will ultimately be applied. A larger field of related organisms are still to be tested (see future work)
From the above it can be seen that an assay has been developed producing two strong bands one specific for L.monocytogenes and the other specific for Listeria species. No amplification products have been found with any other species tested to date other than those that should cause amplification.
Figure 53 shows a list of bacterial species tested to date.
CURRENT POLYMERASE CHAIN REACTION(PCR) METHOD FOR DETECTION OF LISTERIA MONOCYTOGENES IN FOOD SAMPLES WITH KNOWN PRIMERS
1. OBJECTIVE
This current PCR method is based on amplification of certain sequences of DNA on the Listeria monocytogenes genome. It is a multiplex method where two pairs of primers are used in one PCR reaction. One pair of primers is designed specifically for genus Listeria identification and another pair for species monocytogenes identification.
Primer pair for Listeria genus:
Ul 5'- CAG CAG CCG CGG TAA TAC Lane et al (1985)
UII 5 - CTC CAT AAA GGT GAC CCT Stackenbrandt & Curiale (1988)
Primer pair for monocytogenes species:
LMI 5'- CCT AAG ACG CCA ATC GAA Mengaud et al (1988)
LMII 5'- AAG CGC TTG CAA CTG CTC Mengaud et al (1988) Carry out a PCR reaction using the above primers and cycling times of 95°C for 30 seconds, 49°C for 20 seconds and 72°C for 30 seconds, for 29 cycles with a final extension of 72 °C"for 8 minutes.
DNA is extracted from the bacterial cells by heat blasting the cells and adding aliquots to the PCR reaction.
INDUSTRIAL APPLICABILITY
Listeria is a contaminant of food samples and is pathogenic to humans.
The new, specific primers will enable the detection of Listeria and L.monocytogenes in food samples.
Rcoprophilus is a contaminant of water and is also pathogenic to humans.
The invention provides new, specific primers allowing for a simple and convenient assay for its detection. This would enable one to determine whether a sample is polluted with faecal material.
References
Fiksdal L., Maki, J. S., La Croix, S. J. and Staley, J. T. (1985): Survival and detection of Bacteroides spp., prospective indicator bacteria. Applied and Environmental Microbiology. 49: 1, 148-150.
Kreader, C. A. (1995): Design and evaluation of Bacteroides DNA probes for the specific detection of human faecal pollution. Applied and Environmental Microbiology. 61 : 1171-1179.
Mara, D. D. and Oragui, J. I. (1981): Occurrence of Rhodococcus coprophilus and associated actinomycetes in feces, sewage and freshwater . Applied and Environmental Microbiology 42: 1037-1042.
Rainey F. A. et al (1995) Phylogenetic analysis of the genera Rhodococcus and Nocardia and evidence for the evolutionary origin of the genus Nocardia from within the radiation of Rhodococcus species. Microbiology; 141, 523 - 528.
Rainey F. A. et al (1992) 16s rDNA Analysis of Spirochaeta thermophila: Its Phylogenetic Position and Implications for the Systematics of the Order Spirochaetales. System. Appl. Microbiol. 15, 197-202.
Rowbotham, T. J. and Cross, T. (1977): Rhodococcus coprophilus : An aerobic nocardioform actinomycete belonging to the "rhodocrous": complex. Journal of General Microbiology 100: 123- 138.
Bansal N.S. (1996) Development of a polymerase chain reaction for the detection of Listeria monocytogenes in foods. Letters in Applied Microbiology 1996, 22 In press
Border, P. M. et al (1990) Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction. Letters in Applied Microbiology 11, 158-162.
Fluit A. C. et al. (1993) Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay. Applied and Environmental Microbiology May 1289 - 1293
Mengaud, J. et al. (1988) Expression in Escherichia coli and sequence analysis of the listeriolysin O determinants of Listeria monocytogenes. Infection and Immunity 56, 766-772.
Wiedman, M et al (1993) Detection of Listeria monocytogenes with a nonisotopic polymerase chain reaction-coupled ligase chain reaction assy. Applied and Environmental Microbiology Aug. 2743-2745.

Claims

1. A primer which reacts with Listeria monocytogenes but which does not react with related or unrelated species of bacteria.
2. A primer according to claim 1 which is a DNA primer.
3. A primer according to claim 1 or claim 2 which is targeted against the Listeriolysin O gene.
4. A primer according to any one of the preceding claims which is selected from the group comprising 31 OF, 1016R, 715F and 1183R.
5. A primer according to claim 4 which is 31 OF or 1016R.
6. A combination of two primers selected from the group comprising 31 OF, 1016R, 715F and 1183R.
7. The combination of the two primers 31 OF and 1016R.
8. A method for detecting Listeria monocytogenes in a sample comprising the use of a primer or combination of primers according to any one of the preceding claims in a polymerase chain reaction (PCR) method.
9. A primer which reacts with Listeria species but which does not react with related or unrelated species of bacteria.
10. A primer according to claim 9 which is a DNA primer.
11. A primer according to claim 9 or 10 which is targeted against the 23 S rRNA DNA.
12. A primer according to any one of claims 9 to 11 which is selected from the group comprising L318F, L1541F, L1993F, L559R, L2038R and L2534R.
13. A combination of two primers selected from the group comprising L318F, L 154 IF, L1993F, L559R, L2038R and L2534R.
14. A primer according to claim 12 which is L318F or L559R.
15. The combination of the two primers L318F and L559R.
16. A method for detecting Listeria in a sample comprising the use of a primer or combination of primers according to any one of claims 9 to 15 in a polymerase chain reaction method.
17. A method of detecting Listeria monocytogenes in a sample comprising the use of two primers selected from the group comprising 31 OF, 1016R, 715F and 1183R together with two primers selected from the group comprising L318F, L1541F, L1993F, L559R, L2038R and L2534R.
18. A method according to claim 17 in which the four primers are 31 OF, 1016R, L318F and L559R.
19. A primer which reacts with Rhodococcus coprophilus but which does not react with related or unrelated species of bacteria.
20. A primer according to claim 19 which is a DNA primer.
21. A primer according to any one of claims 19 or 20 which is targeted against a 16S rRNA DNA sequence.
22. A primer according to any one of claims 19 to 21 which is selected from the group comprising 143F, 568R, 419F, 443F, 467R and 1124R.
23. A primer according to claim 22 which is 143F or 568R.
24. A combination of two primers selected from the group comprising 143F, 568R, 419F, 443F, 467R and l l24R.
25. The combination of the two primers 143F and 568R.
26. A method of detecting Rhodococcus coprophilus in a sample comprising the use of a primer or combination of primers according to any one of claims 19 to 25 in a PCR method.
27. A method according to claim 8 or claim 16 wherein denaturation is carried out at 92-98┬░C, annealing is carried out at 52-70┬░C and extension is carried out at 65-80┬░C.
28. A method according to claim 26 wherein denaturation is carried out at 92-98┬░C, annealing is carried out at 55-70┬░C and extension is carried out at 65-80┬░C.
29. A method according to claim 27 or 28 wherein the repetitive cycle is carried out 30-50 times.
30. A method according to any one of claims 27-29 wherein the denaturation, annealing and extension steps are carried out for 30-60 seconds each.
PCT/NZ1998/000044 1997-03-27 1998-03-27 DETECTION OF LISTERIA MONOCYTOGENES, LISTERIA SPP., AND $i(RHODOCOCCUS COPROPHILUS) WO1998044153A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU68585/98A AU6858598A (en) 1997-03-27 1998-03-27 Detection of (listeria monocytogenes), (listeria spp.), and (rhodococcus coprophilus)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NZ31450197 1997-03-27
NZ314501 1997-03-27

Publications (1)

Publication Number Publication Date
WO1998044153A1 true WO1998044153A1 (en) 1998-10-08

Family

ID=19926191

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NZ1998/000044 WO1998044153A1 (en) 1997-03-27 1998-03-27 DETECTION OF LISTERIA MONOCYTOGENES, LISTERIA SPP., AND $i(RHODOCOCCUS COPROPHILUS)

Country Status (2)

Country Link
AU (1) AU6858598A (en)
WO (1) WO1998044153A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001068900A2 (en) * 2000-03-15 2001-09-20 Vermicon Ag Method for specifically detecting microorganisms by polymerase chain reaction
CN103571968A (en) * 2013-11-25 2014-02-12 盎亿泰地质微生物技术(北京)有限公司 PCR primer for amplifying rhodococcus and method and kit for detecting rhodococcus
CN105671197A (en) * 2016-04-19 2016-06-15 宁波大学 Detecting method for food-borne pathogenic bacteria listeria monocytogenes
CN109055514A (en) * 2018-04-27 2018-12-21 曾小敏 Single rapid detection method for increasing listeria spp viable bacteria

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0314294A2 (en) * 1987-09-11 1989-05-03 Amoco Corporation Detection of listeria
WO1990008841A1 (en) * 1989-02-06 1990-08-09 Gene-Trak Systems Probes and methods for the detection of listeria
US5523205A (en) * 1988-08-02 1996-06-04 Institut Pasteur DNA probes specific for hemolytic listeria

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0314294A2 (en) * 1987-09-11 1989-05-03 Amoco Corporation Detection of listeria
US5523205A (en) * 1988-08-02 1996-06-04 Institut Pasteur DNA probes specific for hemolytic listeria
WO1990008841A1 (en) * 1989-02-06 1990-08-09 Gene-Trak Systems Probes and methods for the detection of listeria

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 57(9), 1991, THOMAS E.J. et al., "Sensitive and Specific Detection of Listeria Monocytogenes in Milk and Ground Beef with the Polymerase Chain Reaction", pages 2576-2580. *
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 59(9) 1993, BLAIS B. et al., "A Simple RNA Probe System for Analysis of Listeria Monocytogenes Polymerase Chain Reaction Product", pages 2795-2800. *
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Vol. 46(3), 1996, SALLEN B. et al., "Comparative Analysis of 16s and 23s rRNA Sequences of Listeria Species", pages 669-674. *
JOURNAL OF GENERAL MICROBIOLOGY, Vol. 118, 1980, MORDARSKI M. et al., "Ribosomal Ribonucleic Acid Similarities in the Classification of Rhodococcus and Related Taxa", pages 313-319. *
MICROBIOLOGY, Vol. 141, 1995, RAINEY A.R. et al., "Phylogenetic Analysis of the Genera Rhodococcus and Norcardia from Within the Radiation of Rhodococcus Species", pages 523-528. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001068900A2 (en) * 2000-03-15 2001-09-20 Vermicon Ag Method for specifically detecting microorganisms by polymerase chain reaction
WO2001068900A3 (en) * 2000-03-15 2002-08-22 Vermicon Ag Method for specifically detecting microorganisms by polymerase chain reaction
CN103571968A (en) * 2013-11-25 2014-02-12 盎亿泰地质微生物技术(北京)有限公司 PCR primer for amplifying rhodococcus and method and kit for detecting rhodococcus
CN105671197A (en) * 2016-04-19 2016-06-15 宁波大学 Detecting method for food-borne pathogenic bacteria listeria monocytogenes
CN105671197B (en) * 2016-04-19 2019-03-08 宁波大学 A kind of detection method of food-borne pathogens Listeria monocytogenes
CN109055514A (en) * 2018-04-27 2018-12-21 曾小敏 Single rapid detection method for increasing listeria spp viable bacteria

Also Published As

Publication number Publication date
AU6858598A (en) 1998-10-22

Similar Documents

Publication Publication Date Title
Widjojoatmodjo et al. Rapid identification of bacteria by PCR-single-strand conformation polymorphism
Brummer et al. Diversity and seasonal variability of β-Proteobacteria in biofilms of polluted rivers: analysis by temperature gradient gel electrophoresis and cloning
Bhattacharya et al. Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites
Farber An introduction to the hows and whys of molecular typing
Waage et al. Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay
Hoffmaster et al. Molecular subtyping of Bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, United States
Wilson et al. Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: use of the polymerase chain reaction for amplification and detection of staphylococcal enterotoxin genes entB and entC1 and the thermonuclease gene nuc
EP0858514B1 (en) Improved is6110 based molecular detection of mycobacterium tuberculosis
EP1572977A2 (en) Assay and compositions for detection of bacillus anthracis nucleic acid
Uyttendaele et al. Identification of Campylobacter jejuni, Campylobacter coli and Campylobacter lari by the nucleic acid amplification system NASBR
WO2001023608A2 (en) Hybridization probes which specifically detect strains of the genera microbispora, microtetraspora, nonomuria and planobispora
WO1995011996A1 (en) Detection assay for listeria and erwinia microorganisms
US5693467A (en) Mycoplasma polymerase chain reaction testing system using a set of mixed and single sequence primers
JP2540023B2 (en) Detection and identification of mycobacteria
Scheu et al. Rapid detection of Listeria monocytogenes by PCR‐ELISA
Uphoff et al. Detecting Mycoplasma contamination in cell cultures by polymerase chain reaction
Ryu et al. Molecular characterization of Korean Bacillus anthracis isolates by amplified fragment length polymorphism analysis and multilocus variable-number tandem repeat analysis
Nikolakopoulou et al. PCR detection of oxytetracycline resistance genes otr (A) and otr (B) in tetracycline-resistant streptomycete isolates from diverse habitats
EP0669402A2 (en) Nucleic acid sequences specific for mycobacterium kansasii
Bsat et al. A combined modified reverse dot-blot and nested PCR assay for the specific non-radioactive detection of Listeria monocytogenes
Shen et al. Rapid detection and identification of the metabolically diverse genus Gordonia by 16S rRNA-gene-targeted genus-specific primers
CA2255624C (en) Compositions and methods for the detection of mycobacterium kansasii
WO1998044153A1 (en) DETECTION OF LISTERIA MONOCYTOGENES, LISTERIA SPP., AND $i(RHODOCOCCUS COPROPHILUS)
KR100457355B1 (en) Pcr primers for amplifying the gene of pathogenic microorganism, and method and test kit for detecting pathogenic microorganism by using the same
Saint et al. A PCR test for the identification and discrimination of Legionella longbeachae serogroups 1 and 2

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AT AU AZ BA BB BG BR BY CA CH CN CU CZ CZ DE DE DK DK EE EE ES FI FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1998541510

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA