Letters in Applied Microbiology 1990, 11, 158-162
MFS/Q32
Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction P . M . B O R D E RJ,. J . H O W A R D *G, . S . PLASTOW & K . W . SICGENS Dalgety PLC, Group Research Laboratory, Station Road, Cambridge C B l 2 J N , U K Received 1 June 1990 and accepted 7 June 1990
BORDER,P.M., HOWARD,J.J., PLASTOW, G.S. & S I G G E N SK.W. , 1990. Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction. Letters in Applied Microbiology 11, 158-162.
Five oligonucleotide sequences are described that were used as primers in the polymerase chain reaction (PCR) to amplify specific sequences from Listeria DNA. When all five primers were used in combination, three PCR products were possible; a Listeria specific product that occurs with DNA from any Listeria sp., a Listeria monocytogenes specific product that occurs only in the presence of DNA from this organism and a universal product that is found using DNA from any bacterial source. The occurrence of these PCR products was used as a diagnostic test on bacteria isolated from various food samples to detect Listeria sp. and L. mono-
cytogenes.
The detection of the human pathogen Listeria monocytogenes and other members of the genus Listeria in food products by existing methods is a time-consuming procedure, largely because of the need to isolate pure cultures before further characterization may be undertaken. Recent developments in molecular biology have raised the possibility of detecting pathogens in foods and other samples without the need for isolation of pure cultures using target-specific gene probes (see Walker & Dougan 1989; Wernars & Notermans 1990 for recent reviews). Approaches to the detection of Listeria sp. by such methods have used standard hybridization techniques either to detect genes coding for factors thought to be involved in pathogenicity such as a-haemolysin (Datta et al. 1987, 1988), listeriolysin 0 (Mengaud et al. 1988), msp (major secreted polypeptide of L. monocytoyenes, Flamm et al. 1989) and L. monocytogenes DTH factor (Notermans et al. 1989) or to detect specific 16s ribosomal RNA (rRNA) sequences (Klinger et al. 1988; Stackenbrandt &
* Corresponding author.
Curiale 1988). One of the main disadvantages of the methods published to date is that relatively large numbers of target cells need to be present (typically 104-105) in order to yield unambiguous results in a background containing large numbers of non-target micro-organisms (Wernars & Notermans 1990). The recently developed technique of polymerase chain reaction (PCR) using thermostable DNA polymerase (Saiki et al. 1988) permits the rapid amplification of specific DNA sequences by a factor of up to 10’. Polymerase chain reaction methods have been described that will detect low levels of Escherichia coli in clinical samples (Olive 1989), Pseudomonas cepacia in soil samples (Steffan & Atlas 1988), Mycohacterium leprae in armadillo tissues (Hartskeerl et al. 1989) and Aeromonas salmonicida in fish (Barry et al. 1990). The sensitivity of these systems typically approximates to the detection of a single bacterium. To the authors’ knowledge there have been no published reports of DNA amplification using PCR to detect Listeria sp. in food or other samples. The work reported in this paper describes the use of a number of
Detection of Listeria using PCR synthetic oligonucleotide probes as primers in the PCR to amplify DNA sequences from L. monocytogenes and other Listeria sp. Materials and Methods
159
maintained on Tryptose Agar (Difco) and all other strains were maintained on Nutrient Agar (Oxoid). Putative Listeria strains were isolated from a variety of sources using the method of Maclean & Lee (1988).
BACTERIAL STRAINS
P R E P A R A T I O N OF S A M P L E S F O R P C R
The bacterial strains used in this study are detailed in Table 1. All Listeria strains were
DNA from bacteria listed in Table 1 was extracted by the method of Heath et al. (1986). Putative Listeria strains isolated from various sources were used as crude cell lysates in the PCR. Lysates were obtained by resuspending cells in 50 pl of water and heating at 110°C for 5 min.
Table 1. Origin and identity of bacterial strains used Bacterium
Source/reference
Listeria grayi innocua ivanovii monacyt ogenes monorytogenes murrayi seeliyeri welshimeri Jonesia denitrijicans Aeramonas hydrophila Bacillus cereus Brochothrix thermosphacta Erwinia curotovora Escherichia coli K12
NCTC 10815 NCTC 11288 NCTC 11846 NCTC 10357 NCTC 11994 NCTC 10812 NCTC 11856 NCTC 11857 NCTC 10816 NCTC 8049 NCTC 11143 NCTC 10822 SCRI 193 Laboratory strain NM522 NCTC 10243 ATCC 15050
Gemella haemolysans Klebsiella pneumoniae Lactobacillus amylophilus helveticus p 1antarum Salmonella typhimurium Serratia marcescens Staphylococcus aureus Enterococcus faecalis Lactococcus lactis subsp. lactis Veillunellu criceti Yersinia enterocolitica
S Y N T H E S I S OF O L I G O N U C L E O T I D E P R I M E R S
Oligonucleotide primers were synthesized in the Biochemistry Department, University of Leicester on an Applied Biosystems 380B synthesizer using standard protocols. All reagents were supplied by Cruachem, Scotland. Sequences of the oligonucleotide primers used in this study are shown in Table 2. D N A AMPLIFICATION BY PCR
NCIMB 11546 NCIMB 8652 NCIMB 11974 NCIMB 10248 NCIMB 2303 NCIMB 9518 ATCC 8043 NCIMB 6681 ATCC 8043 NCTC 11174
NCTC, National Collection of Type Cultures; NCIMB, National Collection of Industrial and Marine Bacteria; SCRI, Scottish Crops Research Institute; ATCC, American Type Culture Collection.
Polymerase chain reaction was performed on DNA extracts or crude cell lysates essentially as described by Saiki et al. (1988). Reactions were carried out in a final volume of 50 pl, containing 5 pl 10 x PCR buffer (100 mmol/l Tris pH 8.3, 500 mmol/l KCI, 15 mmol/l MgCI,, 0.1% gelatin; all reagents from Sigma), 5 p1 dNTP mix (2 mmol/l each of dGTP, dTTP, dATP and dCTP from Pharmacia Ultrapure dNTP set), 3 pl each appropriate primer (0.1 mg/ml), 0.5 pl AmpliTaq DNA polymerase (Perkin Elmer Cetus) plus 1 pl of appropriate DNA preparation or crude cell lysate. Tubes were overlaid with paraffin oil prior to thermal cycling. Thermal cycling was carried out using a Perkin
Table 2. Sequences of oligonucleotide primers Primer u1 u2 LI1 LM 1 LM2
Derived from or - strand
Sequence (5'-3')
+
CAGCMGCCGCGGTAATWC CCGTCAATTCMTTTRAGTTT CTCCATAAAGGTGACCCT
+
-
+ -
CCTAAGACGCCAATCGAA AAGCGCTTGCAACTGCTC
M denotes A or C; W denotes A or T; R denotes A or G.
Reference Lane et al. (1985) Lane et al. (1985) Stackenbrandt & Curiale (1988) Mengaud et al. (1988) Mengaud et al. (1988)
P . M . Border et al.
160
Elmer Cetus DNA Thermal Cycler. Denaturation, annealing and extension temperatures were 95", 50" and 72°C respectively. Denaturation temperature was maintained for 4 min on the first cycle and for 1 min for the subsequent 29 cycles. Annealing and extension times were maintained at 2 s and 1 min respectively throughout the entire 30 cycles. After 30 cycles an additional extension period of 8 min was maintained. Tubes were then held at 4°C until the PCR products were analysed. Analysis of PCR products was by horizontal agarose gel electrophoresis using 1.5% agarose (SeaKem ME) gels run in Tris (50 mmol/l Sigma), borate (50 mmol/l Sigma), EDTA (2.5 mmol/l BDH) buffer containing 0.1 mg/ml ethidium bromide. Gels were visualized and photographed under U.V. light on a UVP inc. transilluminator.
Results and Discussion Figure 1 shows the PCR products obtained when total genomic DNA from various bacteria was subjected to PCR using a combination of all five primers described in Table 2. The bacteria were selected to include the type strains of all known Listeria sp. plus a wide variety of other bacterial species including some that are phylogenetically closely related to Listeria (e.g. Brochothrix
thermosphacta,
Bacillus
cereus).
The primer sequences were designed to have different specificities. The U1 and U2 primers were
based on 16s rRNA sequences that are essentially conserved throughout all bacteria (Lane et al. 1985). Hence these primers are universal and should yield a product of 408 bp derived from the 16s rRNA genes that are present in genomic DNA from any bacterial source. Reference to Fig. 1 shows that a PCR product of 408 bp was observed for each of the 26 bacterial species used in this study. The LI1 primer was also based on 16s rRNA sequence data (Stackenbrandt & Curiale 1988). When used in conjunction with the U1 primer, the LI1 primer should yield a PCR product of 938 bp. The LI1 primer was designed to be specific to members of the genus Listeria as its sequence is complementary to a 16s rRNA sequence that is highly conserved in all Listeria sp. so far studied but variable in other bacteria (Stackenbrandt & Curiale 1988). Figure 1 shows that the LI1 probe is specific to Listeria sp. since only these samples show the characteristic band at 938 bp. In contrast, none of the other bacterial species tested showed this band amongst their PCR products. As expected Jonesia denitrijicans, formerly called Listeria denitrijicans until its re-classification (Rocourt et al. 1987), does not show a band corresponding to the LIl/Ul product at 938 bp. The LM1 and LM2 primers are based on sequence data of the listeriolysin 0 gene published by Mengaud et al. (1988), and are hence designed to be specific to those bacteria that carry this gene. The results in Fig. 1 show
Fig. 1. Products obtained when genomic DNA from various bacteria were subjected to PCR using a combination of U1, U2, LI1, LM1 and LM2 primers. Lanes 1 and 28, Hae 111 digested pUC 9 standards (587 bp, 458/434 bp and 298 bp); lane 2, Listeria grayi; lane 3, L. innocua; lane 4, L. iuanouii; lane 5, L. monocytogenes NCTC 10357; lane 6, L. monocytogenes NCTC 11994; lane 7, L. murrayi; lane 8, L. seeligeri; lane 9, L. welshimeri; lane 10, Jonesia denitrijicans; lane 11, Aeromonas hydrophila; lane 12, Bacillus cereus; lane 13, Erochothrix thermosphacta; lane 14, Erwinia carotovora; lane 15, Escherichia coli; lane 16, Gemella haemolysans; lane 17, Klebsiella pneumoniue; lane 18, Lactobacillus amylophilus; lane 19, Lact. helueticus; lane 20, Lact. plantarum; lane 21, Sulmonella typhimurium; lane 22, Serratia marcescens; lane 23, Staphylococcus aureus; lane 24, Enterococcus faecalis; lane 25, Lactococcus lactis subsp. luctis; lane 26, Veillonella criceti; lane 27, Yersinza enterocolitica.
Detection of Listeria using P C R that the two L. monocytogenes strains tested were the only bacteria that exhibited the characteristic LMi/LM2 product at 702 bp. This finding suggests that the listeriolysin 0 gene may be unique to L. monocytogenes and that the LMI/LM2 primer combination is therefore specific to this organism. Figure 2 shows the PCR products obtained when various cell lysates from bacteria isolated from a number of sources were subjected to PCR using a combination of all five primers shown in Table 2. Cell lysates prepared from cultures of L. innocua (Fig. 2, lanes 8, 9 and 10) and L. monocytogenes (Fig. 2, NCTC 11994, lanes 11, 12 and 13 and NCTC 10357, lane 18) were also included in this experiment. All isolates tested showed the characteristic U1/U2 PCR product at 408 bp, clearly demonstrating the usefulness of this primer combination as a positive control for the efficiency of the PCR. The cell lysates prepared from known Listeria sp. gave the same PCR products as the corresponding DNA extracts in the first experiment (Fig. 1). In addition to this, several of the unknown isolates showed the characteristic
161
LIl/U1 band to 938 bp (Fig. 2, lanes 3, 5, 14, 16, 17, 23 and 30) indicating the presence of Listeria DNA in the cell lysate. One isolate (Fig. 2, lane 2) also showed the characteristic LMl/LM2 product at 702 bp indicating the presence of L. monocytogenes in the original sample. Subsequent biochemical tests confirmed the results predicted by the PCR method. The results presented in this paper suggest that it may be possible to devise a rapid method for the detection of Listeria sp. in foods and other products based on the PCR. The primer sequences described permit the detection of all members of the genus Listeria so far investigated, as well as the specific detection of L. monocytogenes. We have demonstrated that the method works not only on bacterial DNA extracts but also on crude cell lysates which may be rapidly prepared from culture plates. Although no attempt has been made to determine the detection limits of the system, the sensitivity of PCR-based methods is such that it may prove possible to develop the method into one that will detect Listeria sp. and L. monocytogenes directly in enrichment broths. Such a method would have obvious advantages
Fig. 2. Products obtained when crude cell lysates prepared from bacteria isolated from a variety of food samples were subjected to PCR using a combination of U1, U2, LI1, LM1 and LM2 primers. Lanes 1, 20,21 and 32, Hae I11 digested pUC 9 standard (587 bp, 458/434 bp, 298 bp, 267 bp, 257 bp and 174 bp); lanes 2 , 4 , 5 , 6 , 7 and 17, unknown colonies isolated from wheat; lane 3, unknown colony isolated from cream; lanes 8, 9 and 10, colonies of Listeriu innorua; lanes 1 I, 12 and 13, colonies of L. monocytogenes NCTC 11994; lanes 14, 15 and 16, unknown colonies isolated from potatoes; lane 18, colony of L. rnonocytogenes NCTC 10357; lane 19, unknown colony isolated from cucumber; lanes 22, 23, 26 and 27, unknown colonies isolated from lettuce; lanes 24, 30 and 31, unknown colonies isolated from mushrooms;lanes 25,28 and 29, unknown colonies isolated from radish.
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over existing methods used for the detection of Listeria in foods. The authors would like to thank John Keyte of the University of Leicester, Department of Biochemistry, for synthesizing the oligonucleotides used in this work. References BARRY, T., POWELL,R. & GANNON, F. 1990 A general method to generate DNA probes for microorganisms. Biotechnology 8,233-236. DATA, A.R., WENTZ,B.A. & HILL,W.E. 1987 Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization. Applied and Enuironmental Microbiology 53,225&2259. DATTA,A.R., WENTZ,B.A., SHOCK,D. & TRUCKNESS, M.W. 1988 Synthetic oligodeoxyribonucleotide probes for detection of Listeria monocytogenes. Applied and Environmental Microbiology 54, 29332937. FLAMM, R.K., HINRICHS,D.J. & THOMASHOW, M.F. 1989 Cloning of a gene encoding a major secreted polypeptide of Listeria monocytogenes and its potential use as a species-specific probe. Applied and Enuironmental Microbiology 55,2251-2256. HARTSKEERL, R.A., DEWIT, M.Y.L. & KLATSER,P.R. 1989 Polymerase chain reaction for the detection of Mycobacterium leprae. Journal of General Microbiology 135, 2357-2364. HEATH,L.S., SLOAN,G.L. & HEATH,H.E. 1986 A simple and generally applicable procedure for releasing DNA from bacterial cells. Applied and Enuironmental Microbiology 51, 1138-1 140. KLINCER,J.D., JOHNSON,A., CROAN,D., FLY”, P., WHIPPLE,K., KIMBALL, M., LAWRIE,J. & CURIALE, M. 1988 Comparative studies of nucleic acid hybridization assay for Listeria in foods. Journal of the Association of Analytical Chemistry 71, 669-673. LANE,D.J., PACE, B., OLSEN, G.J., STAHL,D.A., SOGIN, N.R. 1985 Rapid determination of M.L. & PACE, 16s ribosomal RNA sequences for phylogenetic analyses. Proceedings of the National Academy of Science, U S A 82,69554959.
MCCLEAN,D. & LEE, W.H. 1988 Development of USDA/FSIS method for the isolation of Listeria rnonocytogenes from raw meat and poultry. Journal of the Association of Analytical Chemistry 71, 660664. MENGAUD, J., VICENTE, M.F., CHENEVERT, J., PEREIRA, J.M., GEOFFREY, C., GICQUEL-SANZEY, B., BAQUERO, F., PEREZ-DIAZ,J.C.& COSART, P. 1988 Expression in Escherichia coli and sequence analysis of the listeriolysin 0 determinant of Listeria monocytogenes. Infection and Immunity 56, 766772. NOTERMANS,S., CHAKRABORTY, T., LEIMEISTERWACHTER,M., DUFRENNE,J., HEUVELMAN, C.J., MAAS, H., JANSEN,W., WERNARS,K. & GUINEE, P.A.M. 1989. A specific gene probe for detection of bio- and serotyped Listeria strains. Applied and Environmental Microbiology 55,902-906. OLIVE, D.M. 1989 Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase. Journal of Clinical Microbiology 27, 261265. ROCOURT, J., WEHMEYER, U. & STACKBNBRANDT, E. 1987 Transfer of Listeria denitrifcans to Jonesia new-genus as Jonesia denitriJcans newcombination. International Journal of Systematic Bacteriology 37, 266-270. SAIKI,R.K., GELFAND, D.H., STOFFEL, S., SCHARF, S.J., HIGUCHI,R., HORN,G.T., MULLIS,K.B. & ERLICH, H.A. 1988 Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239,4877491. STACKENBRANDT, E. & CURIALE, M. 1988 Detection of Listeria. European Patent Application 88308820.5 STEFFAN, R.J. & ATLAS,R.M. 1988 DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Applied and Environmental Microbiology 54,2185-2191. WALKER, J. & DOUGAN, G. 1989 DNA probes: A new role in diagnostic microbiology. Journal of Applied Bacteriology 67,229-238. WERNERS, K. & NOTERMANS, S. 1990 Gene probes for the detection of food-borne pathogens. In Gene Probes for Bacteria ed. Macario, A.J.L. & de Macario, E.C. pp. 353-388. London: Academic Press.
MFS/Q32
Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction P . M . B O R D E RJ,. J . H O W A R D *G, . S . PLASTOW & K . W . SICGENS Dalgety PLC, Group Research Laboratory, Station Road, Cambridge C B l 2 J N , U K Received 1 June 1990 and accepted 7 June 1990
BORDER,P.M., HOWARD,J.J., PLASTOW, G.S. & S I G G E N SK.W. , 1990. Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction. Letters in Applied Microbiology 11, 158-162.
Five oligonucleotide sequences are described that were used as primers in the polymerase chain reaction (PCR) to amplify specific sequences from Listeria DNA. When all five primers were used in combination, three PCR products were possible; a Listeria specific product that occurs with DNA from any Listeria sp., a Listeria monocytogenes specific product that occurs only in the presence of DNA from this organism and a universal product that is found using DNA from any bacterial source. The occurrence of these PCR products was used as a diagnostic test on bacteria isolated from various food samples to detect Listeria sp. and L. mono-
cytogenes.
The detection of the human pathogen Listeria monocytogenes and other members of the genus Listeria in food products by existing methods is a time-consuming procedure, largely because of the need to isolate pure cultures before further characterization may be undertaken. Recent developments in molecular biology have raised the possibility of detecting pathogens in foods and other samples without the need for isolation of pure cultures using target-specific gene probes (see Walker & Dougan 1989; Wernars & Notermans 1990 for recent reviews). Approaches to the detection of Listeria sp. by such methods have used standard hybridization techniques either to detect genes coding for factors thought to be involved in pathogenicity such as a-haemolysin (Datta et al. 1987, 1988), listeriolysin 0 (Mengaud et al. 1988), msp (major secreted polypeptide of L. monocytoyenes, Flamm et al. 1989) and L. monocytogenes DTH factor (Notermans et al. 1989) or to detect specific 16s ribosomal RNA (rRNA) sequences (Klinger et al. 1988; Stackenbrandt &
* Corresponding author.
Curiale 1988). One of the main disadvantages of the methods published to date is that relatively large numbers of target cells need to be present (typically 104-105) in order to yield unambiguous results in a background containing large numbers of non-target micro-organisms (Wernars & Notermans 1990). The recently developed technique of polymerase chain reaction (PCR) using thermostable DNA polymerase (Saiki et al. 1988) permits the rapid amplification of specific DNA sequences by a factor of up to 10’. Polymerase chain reaction methods have been described that will detect low levels of Escherichia coli in clinical samples (Olive 1989), Pseudomonas cepacia in soil samples (Steffan & Atlas 1988), Mycohacterium leprae in armadillo tissues (Hartskeerl et al. 1989) and Aeromonas salmonicida in fish (Barry et al. 1990). The sensitivity of these systems typically approximates to the detection of a single bacterium. To the authors’ knowledge there have been no published reports of DNA amplification using PCR to detect Listeria sp. in food or other samples. The work reported in this paper describes the use of a number of
Detection of Listeria using PCR synthetic oligonucleotide probes as primers in the PCR to amplify DNA sequences from L. monocytogenes and other Listeria sp. Materials and Methods
159
maintained on Tryptose Agar (Difco) and all other strains were maintained on Nutrient Agar (Oxoid). Putative Listeria strains were isolated from a variety of sources using the method of Maclean & Lee (1988).
BACTERIAL STRAINS
P R E P A R A T I O N OF S A M P L E S F O R P C R
The bacterial strains used in this study are detailed in Table 1. All Listeria strains were
DNA from bacteria listed in Table 1 was extracted by the method of Heath et al. (1986). Putative Listeria strains isolated from various sources were used as crude cell lysates in the PCR. Lysates were obtained by resuspending cells in 50 pl of water and heating at 110°C for 5 min.
Table 1. Origin and identity of bacterial strains used Bacterium
Source/reference
Listeria grayi innocua ivanovii monacyt ogenes monorytogenes murrayi seeliyeri welshimeri Jonesia denitrijicans Aeramonas hydrophila Bacillus cereus Brochothrix thermosphacta Erwinia curotovora Escherichia coli K12
NCTC 10815 NCTC 11288 NCTC 11846 NCTC 10357 NCTC 11994 NCTC 10812 NCTC 11856 NCTC 11857 NCTC 10816 NCTC 8049 NCTC 11143 NCTC 10822 SCRI 193 Laboratory strain NM522 NCTC 10243 ATCC 15050
Gemella haemolysans Klebsiella pneumoniae Lactobacillus amylophilus helveticus p 1antarum Salmonella typhimurium Serratia marcescens Staphylococcus aureus Enterococcus faecalis Lactococcus lactis subsp. lactis Veillunellu criceti Yersinia enterocolitica
S Y N T H E S I S OF O L I G O N U C L E O T I D E P R I M E R S
Oligonucleotide primers were synthesized in the Biochemistry Department, University of Leicester on an Applied Biosystems 380B synthesizer using standard protocols. All reagents were supplied by Cruachem, Scotland. Sequences of the oligonucleotide primers used in this study are shown in Table 2. D N A AMPLIFICATION BY PCR
NCIMB 11546 NCIMB 8652 NCIMB 11974 NCIMB 10248 NCIMB 2303 NCIMB 9518 ATCC 8043 NCIMB 6681 ATCC 8043 NCTC 11174
NCTC, National Collection of Type Cultures; NCIMB, National Collection of Industrial and Marine Bacteria; SCRI, Scottish Crops Research Institute; ATCC, American Type Culture Collection.
Polymerase chain reaction was performed on DNA extracts or crude cell lysates essentially as described by Saiki et al. (1988). Reactions were carried out in a final volume of 50 pl, containing 5 pl 10 x PCR buffer (100 mmol/l Tris pH 8.3, 500 mmol/l KCI, 15 mmol/l MgCI,, 0.1% gelatin; all reagents from Sigma), 5 p1 dNTP mix (2 mmol/l each of dGTP, dTTP, dATP and dCTP from Pharmacia Ultrapure dNTP set), 3 pl each appropriate primer (0.1 mg/ml), 0.5 pl AmpliTaq DNA polymerase (Perkin Elmer Cetus) plus 1 pl of appropriate DNA preparation or crude cell lysate. Tubes were overlaid with paraffin oil prior to thermal cycling. Thermal cycling was carried out using a Perkin
Table 2. Sequences of oligonucleotide primers Primer u1 u2 LI1 LM 1 LM2
Derived from or - strand
Sequence (5'-3')
+
CAGCMGCCGCGGTAATWC CCGTCAATTCMTTTRAGTTT CTCCATAAAGGTGACCCT
+
-
+ -
CCTAAGACGCCAATCGAA AAGCGCTTGCAACTGCTC
M denotes A or C; W denotes A or T; R denotes A or G.
Reference Lane et al. (1985) Lane et al. (1985) Stackenbrandt & Curiale (1988) Mengaud et al. (1988) Mengaud et al. (1988)
P . M . Border et al.
160
Elmer Cetus DNA Thermal Cycler. Denaturation, annealing and extension temperatures were 95", 50" and 72°C respectively. Denaturation temperature was maintained for 4 min on the first cycle and for 1 min for the subsequent 29 cycles. Annealing and extension times were maintained at 2 s and 1 min respectively throughout the entire 30 cycles. After 30 cycles an additional extension period of 8 min was maintained. Tubes were then held at 4°C until the PCR products were analysed. Analysis of PCR products was by horizontal agarose gel electrophoresis using 1.5% agarose (SeaKem ME) gels run in Tris (50 mmol/l Sigma), borate (50 mmol/l Sigma), EDTA (2.5 mmol/l BDH) buffer containing 0.1 mg/ml ethidium bromide. Gels were visualized and photographed under U.V. light on a UVP inc. transilluminator.
Results and Discussion Figure 1 shows the PCR products obtained when total genomic DNA from various bacteria was subjected to PCR using a combination of all five primers described in Table 2. The bacteria were selected to include the type strains of all known Listeria sp. plus a wide variety of other bacterial species including some that are phylogenetically closely related to Listeria (e.g. Brochothrix
thermosphacta,
Bacillus
cereus).
The primer sequences were designed to have different specificities. The U1 and U2 primers were
based on 16s rRNA sequences that are essentially conserved throughout all bacteria (Lane et al. 1985). Hence these primers are universal and should yield a product of 408 bp derived from the 16s rRNA genes that are present in genomic DNA from any bacterial source. Reference to Fig. 1 shows that a PCR product of 408 bp was observed for each of the 26 bacterial species used in this study. The LI1 primer was also based on 16s rRNA sequence data (Stackenbrandt & Curiale 1988). When used in conjunction with the U1 primer, the LI1 primer should yield a PCR product of 938 bp. The LI1 primer was designed to be specific to members of the genus Listeria as its sequence is complementary to a 16s rRNA sequence that is highly conserved in all Listeria sp. so far studied but variable in other bacteria (Stackenbrandt & Curiale 1988). Figure 1 shows that the LI1 probe is specific to Listeria sp. since only these samples show the characteristic band at 938 bp. In contrast, none of the other bacterial species tested showed this band amongst their PCR products. As expected Jonesia denitrijicans, formerly called Listeria denitrijicans until its re-classification (Rocourt et al. 1987), does not show a band corresponding to the LIl/Ul product at 938 bp. The LM1 and LM2 primers are based on sequence data of the listeriolysin 0 gene published by Mengaud et al. (1988), and are hence designed to be specific to those bacteria that carry this gene. The results in Fig. 1 show
Fig. 1. Products obtained when genomic DNA from various bacteria were subjected to PCR using a combination of U1, U2, LI1, LM1 and LM2 primers. Lanes 1 and 28, Hae 111 digested pUC 9 standards (587 bp, 458/434 bp and 298 bp); lane 2, Listeria grayi; lane 3, L. innocua; lane 4, L. iuanouii; lane 5, L. monocytogenes NCTC 10357; lane 6, L. monocytogenes NCTC 11994; lane 7, L. murrayi; lane 8, L. seeligeri; lane 9, L. welshimeri; lane 10, Jonesia denitrijicans; lane 11, Aeromonas hydrophila; lane 12, Bacillus cereus; lane 13, Erochothrix thermosphacta; lane 14, Erwinia carotovora; lane 15, Escherichia coli; lane 16, Gemella haemolysans; lane 17, Klebsiella pneumoniue; lane 18, Lactobacillus amylophilus; lane 19, Lact. helueticus; lane 20, Lact. plantarum; lane 21, Sulmonella typhimurium; lane 22, Serratia marcescens; lane 23, Staphylococcus aureus; lane 24, Enterococcus faecalis; lane 25, Lactococcus lactis subsp. luctis; lane 26, Veillonella criceti; lane 27, Yersinza enterocolitica.
Detection of Listeria using P C R that the two L. monocytogenes strains tested were the only bacteria that exhibited the characteristic LMi/LM2 product at 702 bp. This finding suggests that the listeriolysin 0 gene may be unique to L. monocytogenes and that the LMI/LM2 primer combination is therefore specific to this organism. Figure 2 shows the PCR products obtained when various cell lysates from bacteria isolated from a number of sources were subjected to PCR using a combination of all five primers shown in Table 2. Cell lysates prepared from cultures of L. innocua (Fig. 2, lanes 8, 9 and 10) and L. monocytogenes (Fig. 2, NCTC 11994, lanes 11, 12 and 13 and NCTC 10357, lane 18) were also included in this experiment. All isolates tested showed the characteristic U1/U2 PCR product at 408 bp, clearly demonstrating the usefulness of this primer combination as a positive control for the efficiency of the PCR. The cell lysates prepared from known Listeria sp. gave the same PCR products as the corresponding DNA extracts in the first experiment (Fig. 1). In addition to this, several of the unknown isolates showed the characteristic
161
LIl/U1 band to 938 bp (Fig. 2, lanes 3, 5, 14, 16, 17, 23 and 30) indicating the presence of Listeria DNA in the cell lysate. One isolate (Fig. 2, lane 2) also showed the characteristic LMl/LM2 product at 702 bp indicating the presence of L. monocytogenes in the original sample. Subsequent biochemical tests confirmed the results predicted by the PCR method. The results presented in this paper suggest that it may be possible to devise a rapid method for the detection of Listeria sp. in foods and other products based on the PCR. The primer sequences described permit the detection of all members of the genus Listeria so far investigated, as well as the specific detection of L. monocytogenes. We have demonstrated that the method works not only on bacterial DNA extracts but also on crude cell lysates which may be rapidly prepared from culture plates. Although no attempt has been made to determine the detection limits of the system, the sensitivity of PCR-based methods is such that it may prove possible to develop the method into one that will detect Listeria sp. and L. monocytogenes directly in enrichment broths. Such a method would have obvious advantages
Fig. 2. Products obtained when crude cell lysates prepared from bacteria isolated from a variety of food samples were subjected to PCR using a combination of U1, U2, LI1, LM1 and LM2 primers. Lanes 1, 20,21 and 32, Hae I11 digested pUC 9 standard (587 bp, 458/434 bp, 298 bp, 267 bp, 257 bp and 174 bp); lanes 2 , 4 , 5 , 6 , 7 and 17, unknown colonies isolated from wheat; lane 3, unknown colony isolated from cream; lanes 8, 9 and 10, colonies of Listeriu innorua; lanes 1 I, 12 and 13, colonies of L. monocytogenes NCTC 11994; lanes 14, 15 and 16, unknown colonies isolated from potatoes; lane 18, colony of L. rnonocytogenes NCTC 10357; lane 19, unknown colony isolated from cucumber; lanes 22, 23, 26 and 27, unknown colonies isolated from lettuce; lanes 24, 30 and 31, unknown colonies isolated from mushrooms;lanes 25,28 and 29, unknown colonies isolated from radish.
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