Letters in Applied Microbiology 1992, 15,248-252
Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR) M.A.B. STARBUCK P.J. , H I L L& G . S . A . B . S T E W A R T *University of Nottingham, Faculty of Agriculture and Food Sciences, Department of Applied Biochemistry and Food Science, Sutton Bonington, Loughborough, Leics LEI2 5RD, U K GWGJ163: received 17 June 1992 and accepted 9 July 1992
STARBUCK M.A.B., , H I L L ,P.J. & STEWART, G.S.A.B. 1992. Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR). Letters in Applied Microbiology 15, 248-252. The polymerase chain reaction (PCR) has been used to detect Listeria monocytogenes in whole milk at a level of 0.1 cfu per 30 ml. This high degree of sensitivity has been achieved following enzymatic digestion, polysulphonone membrane filtration and amplification of a nucleotide sequence within the promoter region of ItlyA. Key elements of the procedure are the absence of enrichment culture and a complete solubilization of the membrane filter, ensuring total nucleic acid recovery. The simplicity of the protocol coupled with high sample volumes and exquisite sensitivity extends the relevance of PCR within food and environmental microbiology.
The polymerase chain reaction (PCR) has great potential for the detection of micro-organisms in environmental samples, allowing rapid and specific identification of pathogenic, spoilage and indicator organisms. Many examples of the use of PCR for bacterial detection have been published, for example, in the detection of water-borne pathogens such as Legionella pneumophila (Bej et al. 1991), Shigella and Salmonella (Bej et nl. 1990a), for detection of indicator bacteria such as Escherichia coli in water courses (Bej et al. 1990b), for the detection of viable but nonculturable bacteria (Brauns et a/. 1991) and for the detection of genetically-engineered bacteria in the environment (Steffan 8c Atlas 1988). Other uses of PCR based probes for environmental samples have been recently reviewed (Bej & Mahbubani 1992; Atlas et al. 1992). PCR has also proved very successful in the rapid detection of fastidious pathogens in clinical specimens, including Mycobacterium leprae (Hartskeerl et al. 1989), Clostridium difficile
* Corresponding author.
(Wren et a/. 1990) and pathogenic Treponemes in cerebrospinal fluid (Hay et al. 1990). The application of PCR for bacterial detection within foods has also received much attention, for example the detection of Shigella flexneri in lettuce (Lampel et a/. 1990) and toxigenic E. coli in various foods (Samadpour et al. 1990). By far the majority of studies in this area, however, have targeted the detection of Listeria monocytogenes in milk, cheese and meat products as summarized in Table 1. Without exception these studies have proved the specificity of PCR for the analysis of food samples but have failed to achieve the degree of sensitivity required for pathogen detection in foods. There is no doubt that the PCR technique has the sensitivity and specificity required to achieve the detection limits for bacterial pathogens in food. It is the isolation and harvesting procedures for bacterial target DNA that are the limiting factors to the sensitivity of the assay. In this present work we have therefore developed a novel procedure for bringing milk samples to the PCR reaction which, without
Meat products, vegetables and seafood
10 ml 2% skim milk
0.1 cfu/
to cfu/ Agarose gel electrophoresis
108-103 (depe chees
Agarose gel electrophoresis and identification using oligonucleotide probe hybridization to hylA
Homogenization, cell lysis, centrifugation and precipitation of the aqueous phase with ethanol. Addition schemes employed dialysis, phenol extraction and affinity chromatography 24 h enrichment at 37°C and plated onto Listeria plating medium before DNA extraction using centrifugation, lysis and precipitation 24 h enrichment at 37°C and plated onto Listeria plating medium before DNA extraction using centrifugation, lysis and precipitation. Primary and secondary enrichment cell
0.5 g soft cheese
10 cfu/l
0.1 cfu/
Cells pelleted by centrifugation, washed in PCR buffer and lysed
Artificially contaminated milk
None gi
lo5 cfu/
4-400 c
Detecti L. mon
Agarose gel electrophoresis and identification using oligonucleotide probe hybridization to hlyA
PCR products separated by agarose gel electrophoresis and identified by dot-blot hybridization using the t( or haemolysin sequence PCR products separated by agarose gel electrophoresis and identified by Southern transfer and oligonucleotide hybridization PCR products identified using a 32P labelled oligonucleotide probe specific for Dth 18 gene
Cell lysis of enriched bacterial culture
25 g of ground beef
Hybridization with 32Plabelled oligonucleotide probe Agarose gel electrophoresis
Detection system after PCR amplification
Cell lysis by sonication and boiling with lysozyme and SDS Cells pelleted by centrifugation, saline washed and lysed by microwaving
Summary of DNA preparation method
L. monocytogenes suspension in H,O L. monocytogenes inoculated into whole homogenized milk Cooked sausage
Samde
Table 1. Summary of the literature describing polymerase chain reaction-based detection of Listeria
250
M . A. B. Starbuck et al.
1
GTTTGGTTM TGTCCATFTT ATG? C T W TATAGCZCA? CGTATCXLGT
51
GTACCTGGTA TAGAGAGCGC TGCTAGGTTT GTTGTGTCAG GTAGAGCGGA
101
CATCCATTGT TTTGTAGTTA CAGAGTTCTT TATTGGCTTA TTCCAGTTAT
151
T M G C G M T A TGCTTTTCCG CCTAATGGGA M G T M A M A GTATMAATA
201
AAACAGhGTh A T M M C T A A TGTGCGTTGC AMTAATTCT TATACMAAT
251
GGCCCCCTCC TTTGATTAGT ATATTCCTkT CTTMAGTGA CTTTTATGTT
301
GAGGCATTM CATTTGTTAA CGACGATMA GGGACAGCAG GACTAGAATA
151
MGCTATAAA GCMGCATAT MTATTGCGT TTCATCTTTA G M G C G M T T
401
TCGCCMTAT TATMTTATC MAAGAGAGG GGTGGCAAAC GGTATTTGGC
451
ATTATTAGGT TAAAAAATGTAS&X&-
501
ATGCTAGTTT TTATTACACT TATATTAGTT AGTCTACCM TTGCGCAACA
TA
Fig. 1. The h f y A promoter region of Listeria monocytogenes (Mengaud et af. 1988). The underlined regions represent base pair homology with the PCR primers.
pre-enrichment, provides a detection limit for L. monocytogenes of 0.1 cfu/30 ml (i.e. 10-fold lower than the limit from colony counts).
Materials and Methods BACTERIAL STRAIN A N D MEDIA
For artificial contamination of milk samples Listeria monocytogenes ATCC 23074 (serotype 4b) was used. The strain was grown in Luria Bertani broth (LB) (Maniatis et al. 1982) in shake culture for 14 h at 37°C. Overnight cultures were subcultured in LB and grown to an A,,, of 0.5 prior to use. MILK SAMPLES
UHT, pasteurized or raw milk samples of differing fat quantities were either purchased from local retail sources or obtained from the Faculty dairy. P R E P A R A T I O N OF MILK SAMPLES FOR PCR
Spiked milk samples (30 ml) were vortex mixed for 10 s at high speed; 0.5 ml of 23% (w/v)
tryspin and 3 ml of 0.5% (w/v) Triton X-100 (freshly prepared with, sterile H,O) were then added and samples incubated at 50°C for 20 min (adapted from Pettipher & Rodrigues 1982). During the incubation period 5 ml of prewarmed ( S O T ) 0.5% w/v Triton X-100 was drawn into a sterile disposable syringe and passed through a 13 mm diameter sterile 0.2 pm pore size polysulphonone filter (Supar-200, Gelman). After this pre-treatment the milk sample was passed through the filter to collect bacterial contaminants. If a fatty residue built up on the surface of the filter the milk sample syringe was temporarily removed and the filter treated with pre-warmed surfactant (0.5% (w/v) Triton X-100) before rejoining the sample syringe. The full 30 ml sample was passed through the filter followed by washing with 10 ml of pre-warmed surfactant. Following the filtering procedure the filter was removed aseptically and placed in a sterile 1.5 ml micro-centrifuge tube containing 200 p1 of sterile H,O. The tube was placed at 100°C for 15 min, cooled and 500 p1 of CHCl, added. After a 30 s vortex the filter was completely solubilized. The tube contents were centrifuged briefly (15 000 g, 1 min) and the upper aqueous layer removed completely. The chloroform layer was re-extracted with 200 p1 of H,O and the pooled 1 ml aqueous sample subjected to a standard phenol/chloroform extraction, DNA precipitation by ethanol and vacuum drying of the pellet (Maniatis et al. 1982). PCR TARGET SEQUENCE A N D PRIMERS
The target sequence was the hlyA promoter region (Mengaud et al. 1988) (Fig. 1) 5’ PCR primer (32 mer) 5’ TGT TAT GTC GAC TTA TAG CTC ATC GTA TCA TG 3‘ 3’ PCR primer (54 mer) 5’ CCA AAT TTC ATG GAT CCA GCT GAA TTC TTT T G G GTT TGA GTC TCC TTC TAC 3‘ (The box indicates the hlyA start codon.) The above primers amplify a region of 505 bp and were synthesized using a Milligen Cyclone DNA synthesizer operating on phosphoramidite chemistry.
P C R of Listeria monocytogenes
25 1
without loss. The novel procedure employed here solubilizes the membrane thus avoiding any problem associated with eluting bacteria from the membrane surface. Figure 2 presents the results from a typical PCR amplification of milk samples. We routinely detect L. monocytogenes at levels of 0.1 cfu/30 mi, at least 30-fold better than previous studies and without pre-enrichment. We believe these results demonstrate the importance of sample management in the development of PCR-based assays from foods.
Fig. 2. PCR amplification of UHT milk samples inoculated with Listeria monocytogenes ATCC 23074 using primers defined in the text. Tenfold decreasing numbers of L. monocytogenes are present in samples A-H with lane G having 1 cfu/30 ml. Lane I represents non-inoculated milk and lane J represents milk spiked with sterile distilled water. Hind1 11 digested Lambda DNA is provided in lane K. PCR A S S A Y S A N D D N A A N A L Y S I S
To the dried DNA pellet were added all constituents of the PCR mix and the reaction mix overlaid with 100 p1 of sterile paraffin, the amplification reactions being carried out as described previously (Hill et al. 1991). Following PCR the parafin layer was removed and the aqueous phase washed once with CHC1,. Ten p1 of the aqueous layer was subjected to 0.8% agarose gel electrophoresis as described previously (Stewart et al. 1986). Results and Discussion The strategy to maximize detection of L. monocytogenes in milk was designed to avoid sample pre-enrichment and to minimize the number of steps where target bacterial DNA could be lost. A substantial body of experience has been developed in recent years for the detection of bacteria in milk by direct epifluorescent filter techniques (DEFT). The protocols for such assays require that milk (from full fat to skimmed) be treated to allow efficient filtration. Bacteria captured on the filter are then subjected to fluorescence staining and microscopy. Our initial procedure for rendering milk amenable to filtration was therefore adapted from the DEFT work of Pettipher & Rodrigues (1982). Once filtered, the bacteria present on the filter need to be presented for PCR amplification
P.J. Hill was supported in this study by an AFRC studentship. We thank Sheila Godber for preparing the manuscript. References ATLAS,R.M., SAYLER, G., BURLAGE, R.S. & BEJ,A.K. 1992 Molecular approaches for environmental monitoring of microorganisms. Biotechniques 125, 106-111. BEJ, A.K. & MAHBUBANI, M.H. 1992 Applications of the polymerase chain reaction in environmental microbiology. PCR Methods and Applications 13, 151-1 59. BEJ, A.K., STEFFAN, R.J., DICESARE, J., HAFF, L. & ATLAS,R.M. 1990a Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Applied and Environmental Microbiology 562.307-314. BEJ, A.K., MAHBUBANI, M.H., MILLER,R., DISCESARE, J.L., HAFF,L. & ATLAS, R.M. 1990b Multiplex PCR amplification and immobilised capture probes for detection of bacterial pathogens and indicators in water. Molecular and Cellular Probes 4, 353-365. BEJ, A.K., MAHBUBANI, M.H. & ATLAS,R.M. 1991 Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods. Applied and Environmental Microbiology 572,591-600. BESSESEN, M.T., Luo, Q., ROTBART, H.A., BLASER, M.J. & ELLISON, R.T. 1990 Detection of Listeria monocytogenes by using the polymerase chain reaction. Applied and Environmental Microbiology 569, 29302932. BRAUNS, L.A., HUDSON,M.C. & OLIVER,J.D. 1991 Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnijicans cells. Applied and Environmental Microbiology 57, 2651-2655. FURRER, B., CANDRIAN, U., HOEFELEIN, C. & LUTHY,J. 1991 Detection of Listeria monocytogenes in cooked sausage products and in milk by in uitro amplification of haemolysin gene fragments. Journal oj Applied Bacteriology 70, 312-319. GOLDSTEYN, E.J., KING, R.K., BURCHAK,J. & CANNON, V.P.J.1991 Sensitive and specific detection of Listeria monocytogenes in milk and ground
252
M . A . B. Starbuck et al.
beef with the polymerase chain reaction. Applied and Environmental Microbiology 57,2576-2580. HARTSKEERL, R.A., DEWIT,M.Y.L. & KLATSER, P.A. 1989 Polymerase chain reaction for the detection of Mycobacterium leprae. Journal of General Microbiology 135,2357-2364. HAY,P.E., CLARKE, J.R., STRUGNELL, R.A., TAYLORD. & GOLDMEIER, D. 1990 Use of the ROBINSON, polymerase chain reaction to detect DNA sequences specific to pathogenic treponemes in cerebrospinal fluid. FEMS Microbiology Letters 68, 233-238. HILL,P.J., SWIFT,S. & STEWART, G.S.A.B. 1991 PCR based gene engineering of the Vibrio haroeyi lux operon and the Escherichia trp operon provides for biochemically functional native and fused gene products. Molecular and General Genetics 226,41-48. LAMPEL,K.A., JAGOW,J.A., TRUCKSESS, M. & HILL, W.E. 1990 Polymerase chain reaction for detection of invasive Shigella flexneri in food. Applied and Enoironmental Microbiology 56, 1536-1540. MANIATIS, T., SAMBROOK, J. & FRITSCH,E.F. 1982 Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbour Laboratory Press. MENGAUD, J., VINCENTE, M.F., COSSART, P., CHENEVERT, J., PEREIRA, J.M., GEOFFROY,C., GICQUELB., BANQUERO, F. & PEREZ-DIAZ, J.C. 1988 SANZEY. Expression in Escherichia coli and sequence analysis of the listeriolysin-0 determinant of Listeria monocytogenes. Infection and Immunity 56,766-772. NIEDERHAUSER, C., CANDRIAN, U., HOFELEIN,C.,
JERMINI, M., BUHLER, H.P. & LUTHY,J. 1992 Use of polymerase chain reaction for detection of Listeria monocytogenes in food. Applied and Environmental Microbiology 585, 1564-1568. PETTIPHER, G.L. & RODRIGUES, U.M. 1982 Rapid enumeration of microorganisms in foods by the direct epifluorescent filter technique. Applied and Enuironmental Microbiology 44(4), 809-813. SAMADPOUR, M., LISTON,J., ONGERTH, J.E. & TARR, P.I. 1990 Evaluation of DNA probes for detection of Shiga-like-toxin producing Escherichia coli in food and calf faecal samples. Applied and Enuironmental Microbiology 565, 1212-1215. STEFFAN, R.J. & ATLAS,R.M. 1988 DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Applied and Enoironmental Microbiology 549,2185-2191. STEWART,G.S.A.B., LUBINSKY-MINK, S., JACKSON, C.G., CASSEL,A. & KUHN, J. 1986 pHG165, a pBR322 copy number derivative of pUC8 for cloning and expression. Plasmid 15, 172-181. WERNARS, K., HEUVELMAN, C.J., CHAKRABORTY, T. & NOTERMANS, S.H.W. 1991 Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese. Journal of Applied Bacteriology 70, 121-216. WREN,B.W., CLAYTON, C.L. & TABAQCHALI, S. 1990 Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction. FEMS Microbiology Letters 70, 1-6.
Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR) M.A.B. STARBUCK P.J. , H I L L& G . S . A . B . S T E W A R T *University of Nottingham, Faculty of Agriculture and Food Sciences, Department of Applied Biochemistry and Food Science, Sutton Bonington, Loughborough, Leics LEI2 5RD, U K GWGJ163: received 17 June 1992 and accepted 9 July 1992
STARBUCK M.A.B., , H I L L ,P.J. & STEWART, G.S.A.B. 1992. Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR). Letters in Applied Microbiology 15, 248-252. The polymerase chain reaction (PCR) has been used to detect Listeria monocytogenes in whole milk at a level of 0.1 cfu per 30 ml. This high degree of sensitivity has been achieved following enzymatic digestion, polysulphonone membrane filtration and amplification of a nucleotide sequence within the promoter region of ItlyA. Key elements of the procedure are the absence of enrichment culture and a complete solubilization of the membrane filter, ensuring total nucleic acid recovery. The simplicity of the protocol coupled with high sample volumes and exquisite sensitivity extends the relevance of PCR within food and environmental microbiology.
The polymerase chain reaction (PCR) has great potential for the detection of micro-organisms in environmental samples, allowing rapid and specific identification of pathogenic, spoilage and indicator organisms. Many examples of the use of PCR for bacterial detection have been published, for example, in the detection of water-borne pathogens such as Legionella pneumophila (Bej et al. 1991), Shigella and Salmonella (Bej et nl. 1990a), for detection of indicator bacteria such as Escherichia coli in water courses (Bej et al. 1990b), for the detection of viable but nonculturable bacteria (Brauns et a/. 1991) and for the detection of genetically-engineered bacteria in the environment (Steffan 8c Atlas 1988). Other uses of PCR based probes for environmental samples have been recently reviewed (Bej & Mahbubani 1992; Atlas et al. 1992). PCR has also proved very successful in the rapid detection of fastidious pathogens in clinical specimens, including Mycobacterium leprae (Hartskeerl et al. 1989), Clostridium difficile
* Corresponding author.
(Wren et a/. 1990) and pathogenic Treponemes in cerebrospinal fluid (Hay et al. 1990). The application of PCR for bacterial detection within foods has also received much attention, for example the detection of Shigella flexneri in lettuce (Lampel et a/. 1990) and toxigenic E. coli in various foods (Samadpour et al. 1990). By far the majority of studies in this area, however, have targeted the detection of Listeria monocytogenes in milk, cheese and meat products as summarized in Table 1. Without exception these studies have proved the specificity of PCR for the analysis of food samples but have failed to achieve the degree of sensitivity required for pathogen detection in foods. There is no doubt that the PCR technique has the sensitivity and specificity required to achieve the detection limits for bacterial pathogens in food. It is the isolation and harvesting procedures for bacterial target DNA that are the limiting factors to the sensitivity of the assay. In this present work we have therefore developed a novel procedure for bringing milk samples to the PCR reaction which, without
Meat products, vegetables and seafood
10 ml 2% skim milk
0.1 cfu/
to cfu/ Agarose gel electrophoresis
108-103 (depe chees
Agarose gel electrophoresis and identification using oligonucleotide probe hybridization to hylA
Homogenization, cell lysis, centrifugation and precipitation of the aqueous phase with ethanol. Addition schemes employed dialysis, phenol extraction and affinity chromatography 24 h enrichment at 37°C and plated onto Listeria plating medium before DNA extraction using centrifugation, lysis and precipitation 24 h enrichment at 37°C and plated onto Listeria plating medium before DNA extraction using centrifugation, lysis and precipitation. Primary and secondary enrichment cell
0.5 g soft cheese
10 cfu/l
0.1 cfu/
Cells pelleted by centrifugation, washed in PCR buffer and lysed
Artificially contaminated milk
None gi
lo5 cfu/
4-400 c
Detecti L. mon
Agarose gel electrophoresis and identification using oligonucleotide probe hybridization to hlyA
PCR products separated by agarose gel electrophoresis and identified by dot-blot hybridization using the t( or haemolysin sequence PCR products separated by agarose gel electrophoresis and identified by Southern transfer and oligonucleotide hybridization PCR products identified using a 32P labelled oligonucleotide probe specific for Dth 18 gene
Cell lysis of enriched bacterial culture
25 g of ground beef
Hybridization with 32Plabelled oligonucleotide probe Agarose gel electrophoresis
Detection system after PCR amplification
Cell lysis by sonication and boiling with lysozyme and SDS Cells pelleted by centrifugation, saline washed and lysed by microwaving
Summary of DNA preparation method
L. monocytogenes suspension in H,O L. monocytogenes inoculated into whole homogenized milk Cooked sausage
Samde
Table 1. Summary of the literature describing polymerase chain reaction-based detection of Listeria
250
M . A. B. Starbuck et al.
1
GTTTGGTTM TGTCCATFTT ATG? C T W TATAGCZCA? CGTATCXLGT
51
GTACCTGGTA TAGAGAGCGC TGCTAGGTTT GTTGTGTCAG GTAGAGCGGA
101
CATCCATTGT TTTGTAGTTA CAGAGTTCTT TATTGGCTTA TTCCAGTTAT
151
T M G C G M T A TGCTTTTCCG CCTAATGGGA M G T M A M A GTATMAATA
201
AAACAGhGTh A T M M C T A A TGTGCGTTGC AMTAATTCT TATACMAAT
251
GGCCCCCTCC TTTGATTAGT ATATTCCTkT CTTMAGTGA CTTTTATGTT
301
GAGGCATTM CATTTGTTAA CGACGATMA GGGACAGCAG GACTAGAATA
151
MGCTATAAA GCMGCATAT MTATTGCGT TTCATCTTTA G M G C G M T T
401
TCGCCMTAT TATMTTATC MAAGAGAGG GGTGGCAAAC GGTATTTGGC
451
ATTATTAGGT TAAAAAATGTAS&X&-
501
ATGCTAGTTT TTATTACACT TATATTAGTT AGTCTACCM TTGCGCAACA
TA
Fig. 1. The h f y A promoter region of Listeria monocytogenes (Mengaud et af. 1988). The underlined regions represent base pair homology with the PCR primers.
pre-enrichment, provides a detection limit for L. monocytogenes of 0.1 cfu/30 ml (i.e. 10-fold lower than the limit from colony counts).
Materials and Methods BACTERIAL STRAIN A N D MEDIA
For artificial contamination of milk samples Listeria monocytogenes ATCC 23074 (serotype 4b) was used. The strain was grown in Luria Bertani broth (LB) (Maniatis et al. 1982) in shake culture for 14 h at 37°C. Overnight cultures were subcultured in LB and grown to an A,,, of 0.5 prior to use. MILK SAMPLES
UHT, pasteurized or raw milk samples of differing fat quantities were either purchased from local retail sources or obtained from the Faculty dairy. P R E P A R A T I O N OF MILK SAMPLES FOR PCR
Spiked milk samples (30 ml) were vortex mixed for 10 s at high speed; 0.5 ml of 23% (w/v)
tryspin and 3 ml of 0.5% (w/v) Triton X-100 (freshly prepared with, sterile H,O) were then added and samples incubated at 50°C for 20 min (adapted from Pettipher & Rodrigues 1982). During the incubation period 5 ml of prewarmed ( S O T ) 0.5% w/v Triton X-100 was drawn into a sterile disposable syringe and passed through a 13 mm diameter sterile 0.2 pm pore size polysulphonone filter (Supar-200, Gelman). After this pre-treatment the milk sample was passed through the filter to collect bacterial contaminants. If a fatty residue built up on the surface of the filter the milk sample syringe was temporarily removed and the filter treated with pre-warmed surfactant (0.5% (w/v) Triton X-100) before rejoining the sample syringe. The full 30 ml sample was passed through the filter followed by washing with 10 ml of pre-warmed surfactant. Following the filtering procedure the filter was removed aseptically and placed in a sterile 1.5 ml micro-centrifuge tube containing 200 p1 of sterile H,O. The tube was placed at 100°C for 15 min, cooled and 500 p1 of CHCl, added. After a 30 s vortex the filter was completely solubilized. The tube contents were centrifuged briefly (15 000 g, 1 min) and the upper aqueous layer removed completely. The chloroform layer was re-extracted with 200 p1 of H,O and the pooled 1 ml aqueous sample subjected to a standard phenol/chloroform extraction, DNA precipitation by ethanol and vacuum drying of the pellet (Maniatis et al. 1982). PCR TARGET SEQUENCE A N D PRIMERS
The target sequence was the hlyA promoter region (Mengaud et al. 1988) (Fig. 1) 5’ PCR primer (32 mer) 5’ TGT TAT GTC GAC TTA TAG CTC ATC GTA TCA TG 3‘ 3’ PCR primer (54 mer) 5’ CCA AAT TTC ATG GAT CCA GCT GAA TTC TTT T G G GTT TGA GTC TCC TTC TAC 3‘ (The box indicates the hlyA start codon.) The above primers amplify a region of 505 bp and were synthesized using a Milligen Cyclone DNA synthesizer operating on phosphoramidite chemistry.
P C R of Listeria monocytogenes
25 1
without loss. The novel procedure employed here solubilizes the membrane thus avoiding any problem associated with eluting bacteria from the membrane surface. Figure 2 presents the results from a typical PCR amplification of milk samples. We routinely detect L. monocytogenes at levels of 0.1 cfu/30 mi, at least 30-fold better than previous studies and without pre-enrichment. We believe these results demonstrate the importance of sample management in the development of PCR-based assays from foods.
Fig. 2. PCR amplification of UHT milk samples inoculated with Listeria monocytogenes ATCC 23074 using primers defined in the text. Tenfold decreasing numbers of L. monocytogenes are present in samples A-H with lane G having 1 cfu/30 ml. Lane I represents non-inoculated milk and lane J represents milk spiked with sterile distilled water. Hind1 11 digested Lambda DNA is provided in lane K. PCR A S S A Y S A N D D N A A N A L Y S I S
To the dried DNA pellet were added all constituents of the PCR mix and the reaction mix overlaid with 100 p1 of sterile paraffin, the amplification reactions being carried out as described previously (Hill et al. 1991). Following PCR the parafin layer was removed and the aqueous phase washed once with CHC1,. Ten p1 of the aqueous layer was subjected to 0.8% agarose gel electrophoresis as described previously (Stewart et al. 1986). Results and Discussion The strategy to maximize detection of L. monocytogenes in milk was designed to avoid sample pre-enrichment and to minimize the number of steps where target bacterial DNA could be lost. A substantial body of experience has been developed in recent years for the detection of bacteria in milk by direct epifluorescent filter techniques (DEFT). The protocols for such assays require that milk (from full fat to skimmed) be treated to allow efficient filtration. Bacteria captured on the filter are then subjected to fluorescence staining and microscopy. Our initial procedure for rendering milk amenable to filtration was therefore adapted from the DEFT work of Pettipher & Rodrigues (1982). Once filtered, the bacteria present on the filter need to be presented for PCR amplification
P.J. Hill was supported in this study by an AFRC studentship. We thank Sheila Godber for preparing the manuscript. References ATLAS,R.M., SAYLER, G., BURLAGE, R.S. & BEJ,A.K. 1992 Molecular approaches for environmental monitoring of microorganisms. Biotechniques 125, 106-111. BEJ, A.K. & MAHBUBANI, M.H. 1992 Applications of the polymerase chain reaction in environmental microbiology. PCR Methods and Applications 13, 151-1 59. BEJ, A.K., STEFFAN, R.J., DICESARE, J., HAFF, L. & ATLAS,R.M. 1990a Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Applied and Environmental Microbiology 562.307-314. BEJ, A.K., MAHBUBANI, M.H., MILLER,R., DISCESARE, J.L., HAFF,L. & ATLAS, R.M. 1990b Multiplex PCR amplification and immobilised capture probes for detection of bacterial pathogens and indicators in water. Molecular and Cellular Probes 4, 353-365. BEJ, A.K., MAHBUBANI, M.H. & ATLAS,R.M. 1991 Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods. Applied and Environmental Microbiology 572,591-600. BESSESEN, M.T., Luo, Q., ROTBART, H.A., BLASER, M.J. & ELLISON, R.T. 1990 Detection of Listeria monocytogenes by using the polymerase chain reaction. Applied and Environmental Microbiology 569, 29302932. BRAUNS, L.A., HUDSON,M.C. & OLIVER,J.D. 1991 Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnijicans cells. Applied and Environmental Microbiology 57, 2651-2655. FURRER, B., CANDRIAN, U., HOEFELEIN, C. & LUTHY,J. 1991 Detection of Listeria monocytogenes in cooked sausage products and in milk by in uitro amplification of haemolysin gene fragments. Journal oj Applied Bacteriology 70, 312-319. GOLDSTEYN, E.J., KING, R.K., BURCHAK,J. & CANNON, V.P.J.1991 Sensitive and specific detection of Listeria monocytogenes in milk and ground
252
M . A . B. Starbuck et al.
beef with the polymerase chain reaction. Applied and Environmental Microbiology 57,2576-2580. HARTSKEERL, R.A., DEWIT,M.Y.L. & KLATSER, P.A. 1989 Polymerase chain reaction for the detection of Mycobacterium leprae. Journal of General Microbiology 135,2357-2364. HAY,P.E., CLARKE, J.R., STRUGNELL, R.A., TAYLORD. & GOLDMEIER, D. 1990 Use of the ROBINSON, polymerase chain reaction to detect DNA sequences specific to pathogenic treponemes in cerebrospinal fluid. FEMS Microbiology Letters 68, 233-238. HILL,P.J., SWIFT,S. & STEWART, G.S.A.B. 1991 PCR based gene engineering of the Vibrio haroeyi lux operon and the Escherichia trp operon provides for biochemically functional native and fused gene products. Molecular and General Genetics 226,41-48. LAMPEL,K.A., JAGOW,J.A., TRUCKSESS, M. & HILL, W.E. 1990 Polymerase chain reaction for detection of invasive Shigella flexneri in food. Applied and Enoironmental Microbiology 56, 1536-1540. MANIATIS, T., SAMBROOK, J. & FRITSCH,E.F. 1982 Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbour Laboratory Press. MENGAUD, J., VINCENTE, M.F., COSSART, P., CHENEVERT, J., PEREIRA, J.M., GEOFFROY,C., GICQUELB., BANQUERO, F. & PEREZ-DIAZ, J.C. 1988 SANZEY. Expression in Escherichia coli and sequence analysis of the listeriolysin-0 determinant of Listeria monocytogenes. Infection and Immunity 56,766-772. NIEDERHAUSER, C., CANDRIAN, U., HOFELEIN,C.,
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