A universal protocol for PCR detection of 13 species of foodborne pathogens in foods
Journal of Applied Microbiology1997,83,727–736
A universal protocol for PCR detection of13species of foodborne pathogens in foods
R.-F.Wang,W.-W.Cao and C.E.Cerniglia
Microbiology Division,National Centre for Toxicological Research,FDA,Jefferson,AR,USA
6152/03/97:received14March1997and accepted10April1997
R.-F.WANG,W.-W.CAO AND C.E.CERNIGLIA.1997.A universal protocol for PCR detection
of13species of foodborne pathogens in foods was developed.The protocol used
a universal culture medium and the same PCR conditions with13sets of speci?c
primers.The13species of foodborne pathogens examined were Escherichia coli,E.coli-
ETEC,E.coli-O157:H7,Shigella spp.,Salmonella spp.,Yersinia enterocolitica,Y.
pseudotuberculosis,Vibrio cholerae,V.parahaemolyticus,V.vulni?cus,Listeria
monocytogenes,Staphylococcus aureus and Bacillus cereus.No interference was observed
using the PCR assay when food sample was arti?cially inoculated with each
individual bacterial species.Twelve different seafood samples and two soft cheese
samples without arti?cial inoculation were examined by this protocol.Vibrio
vulni?cus,Salmonella spp.,E.coli,Listeria monocytogenes and Bacillus cereus were
detected in some foods.Internal probe hybridization and nested PCR procedures were used
to con?rm the above?ndings.
INTRODUCTION speci?c pathogenic bacteria from different food materials
(Lantz et al.1994;Hill1996).Numerous papers have been Despite the fact that America’s food supply is the safest in
published on PCR detection of foodborne pathogens includ-the world,tens of millions of cases of foodborne illnesses
ing:Escherichia coli(Bej et al.1990;Victor et al.1991;Wernars occur in the United States every year with a cost to the
et al.1991;Candrian et al.1991;Read et al.1992;Tsai et al. economy of an estimated1–10billion dollars.Therefore,
1993;Jinneman et al.1995);Salmonella(Widjojoatmodjo et the microbiological safety of food has become an important
al.1991;Rahn et al.1992;Cohen et al.1993;Song et al.1993; concern of consumers,industry and regulatory agencies.The Stone et al.1994;Aabo et al.1995;Bulte et al.1995;Cohen U.S.Food and Drug Administration(FDA)gives a high et al.1995);Shigella(Frankel et al.1990;Lampel et al.1990; priority to protecting the public from microbial con-Sethabutr et al.1993;Ra?i et al.1995);Yersinia(Ibrahim et tamination of the food supply(Hill1996).The FDA-con-al.1992;Kwaga et al.1992;Nakajima et al.1992;Rasmussen cerned foodborne bacteria are listed in Bacteriological et al.1995);Vibrio cholerae(Fields et al.1992;Koch et al. Analytical Manual,FDA(FDA1995).These include Esch-1993;Hasan et al.1994;Islam et al.1995;Shangkuan et al. erichia coli,Salmonella,Shigella,Campylobacter,Yersinia1995);V.parahaemolyticus(Tada et al.1992;Lee et al.1995); enterocolitica,Y.pseudotuberculosis,Vibrio cholerae,V.para-V.vulni?cus(Brauns et al.1991;Arias et al.1995);Listeria haemolyticus,V.vulni?cus,Listeria monocytogenes,Staphy-monocytogenes(Wernars et al.1991;Bessesen et al.1991; lococcus aureus,Bacillus cereus,Clostridium perfringens and Cl.Deneer et al.1991;Furrer et al.1991;Thomas et al.1991; botulinum.Wang et al.1991,1992a;Fluit et al.1993;Lawrence et al. Since its introduction in the mid-1980s,polymerase chain1994;Makino et al.1995;Strachan et al.1995;Bassler et al. reaction(PCR)technology has proved to be an invaluable1995;Wiedmann et al.1995;Cano et al.1995;Herman et al. method for the detection of pathogens in food.The PCR1995;Coleman et al.1996;Simon et al.1996),and Staphy-represents a rapid procedure with both high sensitivity and lococcus aureus(Wilson et al.1991;Brakstad et al.1992;Yang speci?city for the immediate detection and identi?cation of et al.1993).
The PCR protocols published for the detection of food-Correspondence to:Carl E.Cerniglia,Microbiology Division,National
borne pathogens are very different;they include the use of Center for Toxicological Research,FDA,Jefferson,AR72079,USA(e-mail:
ccerniglia@https://www.wendangku.net/doc/df729351.html,).different PCR Cycler machines,annealing temperatures,and ?1997The Society for Applied Bacteriology
728R.-F.WANG ET AL.
buffer systems.For routine laboratory analysis,it would be PCR primers
ideal if PCR methods using the same conditions could be
The PCR primers used in this study are listed in Table2. developed.In addition,not all of the published PCR methods
All oligonucleotide primers were synthesized by Universal are very sensitive or speci?c.Some PCR primers have very
DNA Inc.(Tigard,OR,USA)and were unpuri?ed grade. high free energy of the overlapping pentamers at the3?-end
that will cause non-speci?c reactions;some primers have a
stable3?-terminal dimer and some primers have a stable hairpin Food sample preparation and PCR procedures
loop.Therefore,careful choice of the PCR primers,modi-
Food samples were purchased from local food stores.To ?cation of some existing PCR protocols,and development of
ensure a more even distribution,25g of foods were collected new PCR methods for the detection and identi?cation of
from different parts of the food samples,then cut into small a wide range of foodborne bacteria,are necessary for the
pieces under sterile conditions,mixed well,and10g of each development of a routine PCR protocol.
sample inoculated into90ml TSBYE medium.The samples Several problems should be considered when using PCR
were incubated at36°21°C overnight(16–20h)with shak-to detect foodborne pathogens.For example,the complex
ing at150rev min–1.A portion of the sample(0·5ml from composition of food matrices can inhibit the PCR(Lantz et
the upper phase)was mixed with1ml of sterile phosphate-al.1994);PCR cannot distinguish the DNA from live cells
buffered saline(PBS,0·05mol l–1,pH7·4)and centrifuged or dead cells(Hill1996);food particles interfere with the
at9000g for3min.The pellet was washed three times with PCR assay and should be removed before adding to the PCR
PBS,once with water,then resuspended in50m l water.The mixture,because only1–10m l of sample will be subjected to
samples were diluted1:10with1%Triton X-100,incubated the PCR assay.Many procedures have been used to remove
in a boiling water bath for5min,then immediately cooled in the interference and inhibitors,such as DNA isolation and
ice water.A2m l portion of each sample was added to23m l puri?cation methods(Ibrahim et al.1992;Tsai et al.1993;
of PCR mixture containing50mmol l–1Tris-HCl(pH8·5), Song et al.1993;Lantz et al.1994;Bulte et al.1995),enrich-
20mmol l–1KCl,3mmol l–1MgCl2,0·05%bovine serum ment culture with or without DNA isolation steps(Wernars
albumin(BSA,No.A–4378,Sigma Chemical Co.,St Louis, et al.1991;Read et al.1992;Koch et al.1993;Stone et
MO,USA),0·25mmol l–1of each of dATP,dTTP,dCTP, al.1994;Lee et al.1995),and the immunomagnetic beads
dGTP,0·25m mol l–1of each primer,and0·9U of Taq technique(Widjojoatmodjo et al.1991;Fluit et al.1993;Muir
polymerase.The PCR was conducted in a1605Air-Thermal et al.1993).Comparing the results of these studies,it is
Cycler(Idaho Technology,Idaho Falls,ID,USA).The believed that the short enrichment culture procedure without
ampli?cation condition was one cycle of94°C for15s,then DNA isolation is the best approach because it is easy to
35cycles of94°C for3s,50°C for10s and74°C for35s at perform and gives a high sensitivity.In addition,enrichment
the transition speed S-9,and?nally,one cycle of74°C for culture procedures will be helpful for distinguishing the live
2min and45°C for2s.The PCR products(6–10m l of cells from the dead cells,as only the live cells will grow and
each)were separated by electrophoresis in2%agarose gels give positive PCR results.
containing ethidium bromide(1m g ml–1).
In this study,a universal protocol is described which uses
A direct microscope count method(with a Petroff-Hausser the same culture medium and PCR conditions for PCR detec-
counting chamber and a phase-contrast microscope)was used tion of13species of foodborne pathogens in seafood and soft
for determining the bacterial cell numbers in pure culture for cheese samples.
each species as previously described(Wang et al.1994a).
Nested PCR procedures
MATERIALS AND METHODS
PCR conditions were the same as above,but1m l of?rst Bacterial strains,culture medium and growth round PCR products was used for the template and internal conditions primers were used to replace the original primers.
The bacterial strains used in this study and their sources are
given in Table1.Aerobic bacteria were cultured in a universal
Genomic DNA isolation for positive PCR control culture medium,tryptic soy broth yeast extract medium
(TSBYE).The TSBYE contained30g of tryptic soy broth Bacterial cells from pure culture of each species can be used powder with dextrose(Difco Laboratories,Detroit,MI,
to do PCR for positive controls in agarose gel electrophoresis USA),6g of yeast extract,and1l of water.The growth and in this protocol.However,to avoid repeatedly culturing the culture conditions for anaerobic bacteria are the same as those
bacteria,genomic DNA was isolated from these bacteria and previously reported(Wang et al.1994a).used to make PCR products for positive controls.A procedure
?1997The Society for Applied Bacteriology,Journal of Applied Microbiology83,727–736
PCR DETECTION OF FOODBORNE PATHOGENS729 Table1Bacterial strains assayed by PCR in this study
—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
PCR results b
—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Bacterial strains a Eco c ELT O157Shi Sal YE YP VV VIP VC LM SA BC —––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Escherichia coli ATCC25922|????????????E.coli ATCC35401(078:H11)||???????????E.coli ATCC43890(O157:H7)|?|??????????E.coli DH5a|???
E.coli HB101|???
E.coli XL-1|???
Shigella?exneri ATCC12022|??|?????????Sh.?exneri ATCC29903||
Sh.sonnei ATCC25931|??|?????????Sh.sonnei ATCC9290||
Sh.boydii ATCC9207???|??????
Sh.dysenteriae ATCC13313???|??????
Salmonella enteritidis ATCC13076????|????????Salm.typhimurium ATCC14028????|????????Salm.typhimurium4924?|
Yersinia enterocolitica ATCC27729?????|???????Y.pseudotuberculosis ATCC6905??????|??????Y.pseudotuberculosis ATCC29833??|?
Vibrio vulni?cus ATCC27562???????|?????V.parahaemolyticus ATCC17802????????|????V.parahaemolyticus ATCC49398??|
V.cholerae ATCC14035?????????|???Listeria monocytogenes ATCC35152??????????|??L.innocua F4247??????L.grayi ATCC19120??
L.murrayi ATCC25401??
L.welshimeri ATCC35897??
L.ivanovii ATCC19119?????L.seeligeri LA-15??Staphylococcus aureus ATCC25923???????????|?Staph.xylosus ATCC29971??
Staph.epidermidis ATCC12228?????????????Bacillus cereus ATCC11778????????????|B.cereus4AC?|B.subtilis ATCC6051?????????????B.stearothermophilis ATCC7953??B.circulans??—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––a In addition to the bacterial strains listed in this table,we also obtained negative PCR results for all above13PCR primer sets for the following bacterial species:Klebsiella pneumoniae ATCC13883,Pseudomonas aeruginosa ATCC27853,Citrobacter freundii ATCC8090, Enterococcus faecalis ATCC19433,Corynebacterium kutscheri ATCC15677,Streptococcus bovis ATCC9809,Proteus vulgaris ATCC 13315,Pasteurella pneumotropica ATCC35149,Campylobacter jejuni ATCC35918,Mycoplasma gallisepticum ATCC5969,Micrococcus luteus ATCC4698,Bacteroides distasonis ATCC8503,Bacteroides thetaiotaomicron ATCC29148,Bacteroides vulgatus ATCC8482,
Bi?dobacterium adolescentis ATCC15703,Clostridium clostridiiforme ATCC29084,Clostridium perfringens ATCC3626,Eubacterium limosum ATCC8486,Eubacterium biforme ATCC27806,Fusobacterium prausnitzii ATCC27768,Peptostreptococcus productus ATCC27340, Lactobacillus acidophilus ATCC332,and Propionibacterium acnes ATCC6919.b PCR results:|,positive;?,negative;blank,not determined.
c Abbreviations for PCR against the bacteria:Eco,Escherichia coli;ELT,E.coli-ETEC;O157,E.coli-O157:H7;Shi,Shigella spp.;
Sal,Salmonella spp.;YE,Yersinia enterocolitica;YP,Y.pseudotuberculosis;VV,Vibrio vulni?cus;VIP,V.parahaemolyticus;VC,V.cholerae; LM,Listeria monocytogenes;SA,Staphylococcus aureus;BC,Bacillus cereus.
?1997The Society for Applied Bacteriology,Journal of Applied Microbiology83,727–736
730R.-F.WANG ET AL.
Table2PCR primers and the PCR sensitivities(minimum cell numbers in the assay for positive PCR results)
—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Product Sensitivity
Species Target gene PCR primers’sequences(5?-3?)size(cells)Reference
—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Escherichia coli malB Eco-1,GACCTCGGTTTAGTTCACAGA585bp20Candrian et al.
promoter Eco-2,CACACGCTGACGCTGACCA(1991)
E.coli-ETEC enterotoxin(LT)EcoLT-1,117bp40Victor et al.
gene GAGACCGGTATTACAGAAATC(1991)
EcoLT-2,GAGGTGCATGATGAATCCAG
E.coli-O157:H7hlyA gene O157–3,GTAGGGAAGCGAACAGAG361bp2This study
O157–4,AAGCTCCGTGTGCCTGAA
Shigella spp.ipah gene Shi-1,CTTGACCGCCTTTCCGATAC610bp5×104Sethabutr et al.
Shi-2,CAGCCACCCTCTGAGAGTA(1993) Salmonella spp.invA gene Sal-3,TATCGCCACGTTCGGGCAA275bp40Rahn et al.
Sal-4,TCGCACCGTCAAAGGAACC(1992) Yersinia enterocolitica enterotoxin gene YE-1,CTGTCTTCATTTGGAGCATTC159bp4Ibrahim et al.
YE-2,GCAACATACATCGCAGCAATC(1992) Yersinia pseudotuberculosis inv gene YP-3,CTTGGCTGATGGCACGAT440bp40This study
YP-4,TCGTCACCTGACCCTGAT
Vibrio cholerae toxin gene VC-1,GGCAGATTCTAGACCTCCT563bp40Fields et al.
VC-2,TCGATGATCTTGGAGCATTC1992
Vibrio vulni?cus cytolysin gene VV–1,CTCACTGGGGCAGTGGCT383bp100Brauns et al.
VV–2,CCAGCCGTTAACCGAACCA1991
Vibrio parahaemolyticus genomic DNA VIP-1,GAATTCGATAGGGTGTTAACC381bp4Lee et al.(1995)
VIP-2,ATCCTTGAACATACGCAGC
Staphylococcus aureus nuclease gene SA-1,GCGATTGATGGTGATACGGTT276bp20Brakstad et al.
SA-2,CAAGCCTTGACGAACTAAAGC(1992)
Listeria monocytogenes hemolysin gene LM-1,CGGAGGTTCCGCAAAAGATG234bp20Furrer et al.
LM-2,CCTCCAGAGTGATCGATGTT(1991) Bacillus cereus hemolysin gene BC-1,CTGTAGCGAATCGTACGTATC185bp500This study
BC-2,TACTGCTCCAGCCACATTAC
—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––modi?ed from Easy-DNA kit(Invitrogen Co.,San Diego,in100m l of TE buffer(10mmol l–1Tris,1mmol l–1EDTA). CA,USA)was used for isolation of genomic DNA from all
The isolated genomic DNA was diluted with1%Triton X-13bacterial species.Brie?y,1ml of an overnight culture of100to about10ng m l–1.The diluted DNA(1–2m l)was
directly added to23m l of PCR mixture and PCR was done the bacterial cells was centrifuged,washed once with PBS.
Solution A(supplied by the kit),350m l,was added to the as described before.
pellet and mixed well;the mixture was heated at65°C for
10min for most of the bacterial species but for60min for
Listeria monocytogenes,Staphylococcus aureus and Bacillus Internal probe hybridization
cereus.A2m l aliquot of RNase(2mg ml–1)was added to the
tube and heated at65°C for5min,then150m l of solution B Five internal probes were designed:Salm-pb for Salmonella, was added and mixed well.Chloroform(500m l)was added
Eco-pb for E.coli,VV-pb for Vibrio vulni?cus,LM-pb for and mixed well,then centrifuged at14000g for10min.The Listeria monocytogenes and BC-pb for Bacillus cereus.The
primers’sequences(5-3)are:Salm-pb,TTGTCACCGT upper phase was transferred to1ml100%ethanol and mixed
well,then cooled at–20°C for20min,and centrifuged for GGTCCAGTT;Eco-pb,TCTCCTGATGACGCA 10min at14000g.The pellet was washed with75%ethanol
TAGTC;VV-pb,CTTCAAACCGAACTATGACGT; and dried.The genomic DNA in the pellet was resuspended BC-pb,GGAGCTGTACAACTTGCCA;LM-pb,AACCT
?1997The Society for Applied Bacteriology,Journal of Applied Microbiology83,727–736
PCR DETECTION OF FOODBORNE PATHOGENS731 CGGAGACTTACGAGA.The internal probes were also results are presented in Table1.Only E.coli primer set(Eco-used for the nested PCR primers.
1and Eco-2)cross reacts with Shigella?exneri and Sh.sonnei; 32P-labelled internal probes were obtained by using Ter-all other PCR methods are speci?c to the target species.It
should also be noted that except for the species listed in Table minal Deoxynucleotidyl Transferase(TdT)enzyme(Stra-
tagene,La Jolla,CA,USA)with32P-dCTP.A method1,23species also tested negative with all of the13PCR modi?ed from that of Rosenberg et al.(1990)was used to
methods.These bacteria are listed in the legend of Table1. achieve higher labelling ef?ciencies.Brie?y,1m l(20ng)of The PCR products for the13species are shown in Fig.1. each oligomer probe was added to5m l of32P-dCTP and2m l
The sizes of PCR products are the same as predicted(Table of5×CoCl2buffer(supplied in the kit).The tube was2).Some extra bands above383bp may be produced when
using a high concentration of cells or DNA of Vibrio vulni?cus incubated at65°C for10min and cooled in ice-water.TdT
enzyme(2m l,13U m l–1)was added to the mixture and(lane12,Fig.1);however,there are no cross-reactions with incubated at37°C for60min;0·5mol l–1EDTA was then
the other species tested(Table1).
added to stop the reaction.The crude labelled probe was then The PCR sensitivities for pure culture of each species were used in DNA hybridization without puri?cation.
determined and are listed in Table2.The PCR sensitivities The PCR products with the loading dye were heated at
100°C for5min,and then immediately cooled in ice-water
to denature the DNA;l m l of each denatured PCR product
was spotted onto a sheet of nitrocellulose membrane(NC)
with pipette tips,and then dried in vacuum at80°C for
30min.Before hybridization,the membrane was given a
treatment in prehybridization buffer consisting of
5×Denhardt(1×Denhardt is0·02%Ficoll,0·02%poly-
vinylpyrrolidone,0·02%bovine serum albumin),0·1%SDS
and6×STE(10×STE is0·1mol l–1Tris,pH8·0,1mol
l–1NaCl,10mmol l–1EDTA,pH8·0),plus100m g ml–1
denatured salmon sperm DNA and held at55°C for4h.The
NC membranes were then placed into separate15ml tubes
each containing5ml of the prehybridization buffer plus each
of the total11m l of the32P-labelled probes.The tubes were
shaken at50°C overnight.After hybridization,the mem-
branes were washed three times for5min each in2×SSC–
0·1%SDS solution at room temperature,then washed three
to six times for20min each in1×SSC–0·1%SDS at50°C.
Finally,the membranes were washed in1×SSC without
SDS and mounted in a cassette with intensifying screen and
X-ray?lm and exposed at–70°C for0·5–2h.
RESULTS
Fig.1PCR results by the corresponding primer sets for the Development of the PCR procedures
single bacterial species inoculated into shrimp.The
PCR procedures for the detection of13bacterial species
procedures were described in the text.The PCR products were
separated in a2%agarose https://www.wendangku.net/doc/df729351.html,nes:m,DNA size marker;1,YE-were developed in this study.Results of the PCR assays are
1/YE-2,Yersinia enterocolitica ATCC27729;2,YP-3/YP-4, summarized in Table1.The primer’s sequence,the target,
Yersinia pseudotuberculosis ATCC6905;3,Eco-1/Eco-2,Escherichia the PCR product’s size,and the references,are listed in Table
coli ATCC25922;4,EcoLT-1/EcoLT-2,E.coli(078:H11)
2.New PCR methods for Bacillus cereus,Yersinia
ATCC35401;5,O157–3/O157–4,E.coli(O157:H7)ATCC pseudotuberculosis and Escherichia coli-O157:H7were de-
43890;6,LM-1/LM-2,Listeria monocytogenes ATCC35152;7, veloped in this study.PCR primers for the other10species
SA-1/SA-2,Staphylococcus aureus ATCC25923;8,Sal-
are modi?ed from earlier publications.The purpose of modi-3/Sal-4,Salmonella enteritidis ATCC13076;9,Shi-1/Shi-2,
?cation of the primers was to make all13PCR methods Shigella?exneri ATCC12022;10,VC-1/VC-2,Vibrio cholerae suitable for the same PCR conditions,such as the same ATCC14035;11,VIP-1/VIP-2,Vibrio parahaemolyticus ATCC annealing temperature of50°C in Idaho Air-Thermal Cycler.17802;12,VV-1/VV-2,Vibrio vulni?cus ATCC27562;13,BC-The speci?cities of the13PCR methods were tested and the
1/BC-2,Bacillus cereus ATCC11778
?1997The Society for Applied Bacteriology,Journal of Applied Microbiology83,727–736
732R.-F.WANG ET AL.
Fig.2PCR examination of foodborne pathogens in food samples.Panel A,the PCR products in a2%agarose gel.Panel B,internal
probe hybridization with each of the corresponding internal probe for con?rmation of the PCR https://www.wendangku.net/doc/df729351.html,ne m,DNA size
marker;Lanes1–3,PCR for E.coli from3samples:lane1,DNA of E.coli for positive control,lane2,Mahi-mahi?sh?llets,lane3,
Cape Capensis?sh;Lanes4–5,PCR for Listeria monocytogene from2samples:lane4,DNA of L.monocytogene for positive control,lane5, Mahi-mahi?sh?llets;Lanes6–8,PCR for Bacillus cereus from3samples:lane6,DNA of B.cereus for positive control,lane7,shrimp, lane8,Neufchatel spreadable cheese;Lanes9–12,PCR for Salmonella from4samples:lane9,DNA of Salm.enteritidis for positive control, lane10,Flounder,lane11,fresh Cat?sh nuggets,lane12,Neufchatel spreadable cheese;Lanes13–17,PCR for Vibrio vulni?cus from
5samples:lane13,DNA of V.vulni?cus for positive control,lane14,Snow Crab clusters,lane15,fresh Salmon?sh?llets,lane16,Cape Capensis?sh,lane17,Neufchatel spreadable cheese
Table3PCR detection of13food-borne pathogenic bacterial species in12seafood samples and two soft cheese samples
—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––PCR primers a and PCR results b
—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Food samples YE a YP ECO EcoLT O157LM SA Sal Shi VC VIP VV BC —––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Cooked cray?sh?b????????????Snow crab clusters???????????|?Salmon?sh?llets???????????|?Flounder???????|?????Fresh bay scallops?????????????Oysters?????????????Fresh cat?sh nuggets???????|?????Shrimp(medium)?????????????Mahi-mahi?sh?llets??|??|???????Cape capensis?sh??|????????|?Shrimp(small)????????????|Oysters?????????????Italian cream cheese?????????????Neufchatel spreadable cheese???????|???||—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––a PCR primers:YE,Yersinia enterocolitica.YP,Yersinia pseudotuberculosis.ECO,Escherichia coli.EcoLT,ETEC-E.coli.O157,O157:H7-E.coli.LM,Listeria monocytogenes.SA,Staphylococcus aureus.Sal,Salmonella enteritidis.Shi,Shigella.VC,Vibrio cholerae.VIP,
Vibrio parahaemolyticus.VV,Vibrio vulni?cus.BC,Bacillus cereus.
b PCR results:|,PCR positive;?,PCR negative.
?1997The Society for Applied Bacteriology,Journal of Applied Microbiology83,727–736
PCR DETECTION OF FOODBORNE PATHOGENS733 were20,40,2,5×104,40,4,40,40,4,100,20,20and500described in this study,the bacterial mixture should not cells in the assay system for E.coli,E.coli-ETEC,E.coli-
interfere with the PCR detection of a speci?c species(Wang O157:H7,Shigella spp.,Salmonella spp.,Yersinia enter-et al.1994a,1994b).Particularly,if the detected species is ocolitica,Y.pseudotuberculosis,Vibrio cholerae,V.par-
more predominant,the PCR will not give a non-speci?c ahaemolyticus,V.vulni?cus,Listeria monocytogenes,Staph.reaction.Figure1provides evidence that when100cells of aureus and Bacillus cereus,respectively.
each species were separately added to a food sample following
overnight culture,the PCR did not give non-speci?c reactions
or interferences.The speci?city was obtained by using an Use of the PCR procedures for examination of the
Idaho Air Thermo-Cycler machine which could resolve most 13bacterial species in seafood samples
of the non-speci?c products(Wang et al.1995).Another In order to determine whether the universal culture medium,
reason for using the Idaho Cycler is the detection time;the TSBYE,would be suitable for all13bacterial species,100Idaho Cycler takes less than30min,while other PCR Cyclers cells of each single bacterial species were separately inoculated
need2–5h.
onto10g of food sample.Each sample was added to90ml of In the PCR detection of food samples which were not TSBYE and incubated at36°21°C overnight(16–20h)
arti?cially inoculated with any speci?c bacterial species,mul-with shaking at150rev min–1.A portion of the sample(0·5ml tiple DNA bands may be observed.If the DNA band could from the upper phase)was taken for PCR assay using the
not distinguish the correct PCR product’s size from the non-procedure described in the methods section.All of the13speci?c DNA bands,a second gel was needed to run with a bacterial species are suitable for culture in this TSBYE med-
standard PCR product for that species.The standard PCR ium and the procedure works well(Fig.1).No interference products for all13species could be made from pure culture
of each bacterial species,or from the bacterial genomic DNA was observed.The PCR products from the inoculated sam-
ples shown in agarose gel are the same as those produced by of each species.A simple and reliable genomic DNA isolation the bacterial cells in pure culture,or produced by the isolated
procedure is reported which can make genomic DNA from bacterial genomic DNA.1ml culture of each bacterial species;the DNA can be stored Actual examinations of the bacterial contamination in foods
at–20°C for long-term use.
by the PCR assay were conducted for12seafood samples and Internal probe hybridization was used to con?rm the PCR two soft cheese samples;the total results are presented in
results for the food samples(Panel B in Fig.2).The Salm-Table3.The positive PCR results from the food samples are pb and VV-pb gave strong hybridization capability.These shown in Panel A of Fig.2.Escherichia coli was detected in
two probes were washed six times for20min each in two seafood samples,L.monocytogenes was detected in one1×SSC–0·1%SDS at50°C,but only three times for seafood sample,Salmonella was detected in two seafood sam-
the other three probes.During the washing steps,a Geiger ples and one soft cheese sample,B.cereus was detected in one Counter was used to monitor the radioactivity on the NC seafood sample and one cheese sample,and V.vulni?cus was
membranes and to determine the washing strength.This detected in three seafood samples and one cheese sample.monitoring makes the radioactive probe hybridization better
than non-radioactive probe hybridization.However,the non-Panel B of Fig.2shows the con?rmation result by internal
probe hybridization.All positive PCR products were hybrid-radioactive probe hybridization could also be used for the ized with each of the internal probes.Nested PCR procedures
con?rmation if safe laboratory handling of radioactive were also used to con?rm the positive PCR results and all material is a concern.
positive PCR products were positive in nested PCR(data not
Nested PCR is rapid and easy to perform for the con-shown).The nested PCR procedures were conducted using:?rmation but great care must be taken to avoid carry-over Eco-pb|Eco-2for E.coli(the PCR product is499bp);
contamination when pipetting the?rst round of PCR prod-LM-pb|LM-2for L.monocytogenes(the PCR product is ucts.
194bp);Salm-pb|Salm-4for Salmonella(the PCR product
PCR were also performed for homogenized and?ltered is228bp);BC-pb|BC-2for B.cereus(the PCR product is food samples,but positive PCR results were not obtained as
the complex composition of food matrices inhibited the PCR. 162bp);and VV-pb|VV-2for V.vulni?cus(the PCR prod-
uct is322bp).In previously developed PCR methods(Wang et al.1991,
1992a,1992b,1993a,1993b,1994a,1994b,1996a,1996b,
1996c;Slavik et al.1993),the PCR sensitivity for pure culture DISCUSSION
of bacteria could reach to two cells in the reaction mixture, Food,if it is microbiologically contaminated,may contain
and the PCR sensitivities for most bacterial species were4–multiple bacterial species.After culture of the food samples100cells.In this study,only one PCR method(Shigella)is in
low sensitivity(5×104cells),i.e.about5×107cells of Shi-with non-selective medium TSBYE,many different bacterial
species will grow.In most cases,using the PCR protocol gella in1ml culture might be needed to produce PCR positive ?1997The Society for Applied Bacteriology,Journal of Applied Microbiology83,727–736
734R.-F.WANG ET AL.
enterotoxigenic strains by primer-directed enzymatic ampli-results.However,the PCR sensitivity in this protocol should
?cation of speci?c sequences.International Journal of Food Micro-be enough because in most cases,the bacterial cells in over-
biology12,339–352.
night culture can reach109–1010cells ml–1.
Cano,R.J.,Norton,D.M.,Inzunza,A.E.,Sanchez,J.G.and Oste, Shorter culture time such as6h should be enough for
C.(1995)Polymerase chain reaction assay coupled with?u-most of the species.However,overnight culture is convenient
orescence detection on microwell plates for Listeria monocytogenes for the8h working schedule.
in foods.Journal of Food Protection58,614–620.
Boiling the bacterial cells in1%Triton X-100,then Cohen,N.D.,Neibergs,H.L.,McGruder,E.D.et al.(1993)Genus-immediately cooling in ice-water,was ef?cient for releasing speci?c detection of Salmonellae using the polymerase chain reac-and denaturing the DNA template.The resulting materials tion(PCR).Journal of Veterinary Diagnosis and Investigation5, were directly subjected to the PCR assay without DNA iso-368–371.
lation and puri?cation steps.This procedure has worked well
Cohen,N.D.,Wallis,D.E.,Neibergs,H.L.and Hargis,B.M.(1995)
Detection of Salmonella enteritidis in equine feces using the poly-for a wide phylogenetic spectrum of bacteria and viruses
merase chain reaction and genus-speci?c oligonucleotide primers. (Wang et al.1991–1996).
Journal of Veterinary Diagnosis and Investigation7,219–222. The major contribution of this study is the development
Coleman,S.S.,Melanson,D.M.,Biosca,E.G.and Oliver,J.D. of a protocol using a universal culture medium and the same
(1996)Detection of Vibrio vulni?cus biotypes1and2in eels PCR conditions for PCR detection of more than a dozen
and oysters by PCR ampli?cation.Applied and Environmental foodborne bacterial species in foods.PCR procedures for
Microbiology62,1378–1382.
other pathogenic E.coli,or other non-anaerobic foodborne Deneer,H.G.and Boychuk,I.(1991)Species-speci?c detection of pathogenic bacterial species may also be incorporated into L.monocytogenes by DNA ampli?cation.Applied and Environ-this protocol.The protocol could be developed as a routine mental Microbiology57,606–609.
procedure for foodborne pathogen detection.Fields,P.I.,Popovic,T.,Wachsmuth,K.and Olsvik,O.(1992)Use
of polymerase chain reaction for detection of toxigenic Vibrio
cholerae01strains from the Latin American cholera epidemic.
Journal of Clinical Microbiology30,2118–2121. REFERENCES
Fluit,A.C.,Torensma,R.,Visser,M.J.C.et al.(1993)Detection of Aabo,S.,Andersen,J.K.and Olsen,J.E.(1995)Research note-Listeria monocytogenes in cheese with the magnetic immuno-PCR detection of Salmonella in minced meat by the polymerase chain assay.Applied and Environmental Microbiology59,1289–1293. reaction method.Letters in Applied Microbiology21,180–182.Food and Drug Administration(1992,1995)Bacteriological Ana-Arias,C.R.,Garay,E.and Aznar,R.(1995)Nested PCR method lytical Manual,7th and8th edn.Arlington,VA:AOAC Inter-for rapid and sensitive detection of Vibrio vulni?cus in?sh,sedi-national.
ments,and water.Applied and Environmental Microbiology61,Frankel,G.,Riley,L.and Giron,J.A.(1990)Detection of Shigella 3476–3478.in feces using DNA ampli?cation.Journal of Infectious Diseases Bassler,H.A.,Flood,S.J.A.,Livak,K.J.,Marmaro,J.,Knorr,R.161,1252–1256.
and Batt,C.A.(1995)Use of a?uorogenic probe in a PCR-based Furrer,B.,Candrian,U.,Hoefelein,C.and Luethy,J.(1991)Detec-assay for the detection of Listeria monocytogenes.Applied and tion and identi?cation of Listeria monocytogenes in cooked sausage Environmental Microbiology61,3724–3728.products and in milk by in vitro ampli?cation of haemolysin gene Bej,A.K.,Steffan,R.J.,DiCesare,J.,Haff,L.and Atlas,R.M.fragments.J ournal of Applied Bacteriology70,372–379. (1990)Detection of coliform bacteria in water by polymerase Hasan,J.A.K.,Chowdhury,M.A.R.,Shahabuddin,M.,Huq,A., chain reaction and gene probes.Applied and Environmental Micro-Loomis,L.and Colwell,R.R.(1994)Cholera toxin gene poly-biology56,307–314.merase chain reaction for detection of non-culturable Bessesen,M.T.,Luo,Q.,Rotbart,H.A.,Blaser,M.J.and Noter-Vibrio cholerae01.World Journal of Microbiology and Biotechnology mans,S.H.W.(1991)Detection of Listeria monocytogenes by using10,568–571.
the PCR.Applied and Environmental Microbiology56,2930–2932.Herman,L.M.F.,Deblock,J.H.G.E.and Moermans,R.J.B.(1995) Brakstad,O.G.,Aasbakk,K.and Maeland,J.A.(1992)Detection of Direct detection of Listeria monocytogenes in25milliliters of raw Staphylococcus aureus by polymerase chain reaction ampli?cation milk by a two-step PCR with nested primers.Applied and Environ-of the nuc gene.Journal of Clinical Microbiology30,1654–1660.mental Microbiology61,817–819.
Brauns,L.A.,Hudson,M.C.and Oliver,J.D.(1991)Use of the Hill,W.E.(1996)The polymerase chain reaction:applications for polymerase chain reaction in detection of culturable and non-the detection of foodborne pathogens.Critical Reviews in Food culturable Vibrio vulni?cus cells.Applied and Environmental Micro-Science and Nutrition36,123–173.
biology57,2651–2655.Ibrahim,A.,Liesack,W.and Stackebrandt,E.(1992)Polymerase Bulte,M.and Jakob,P.(1995)The use of a PCR-generated INVA chain reaction-gene probe detection system speci?c for patho-probe for the detection of Salmonella spp.in arti?cially and genic strains of Yersinia enterocolitica.Journal of Clinical Micro-naturally contaminated foods.International Journal of Molecular biology30,1942–1947.
Microbiology26,335–344.Islam,M.S.,Alam,M.J.,Miah,M.A.,Felsenstein,A.and Sack, Candrian,U.,Furrer,B.,Hofelein,C.,Meyer,R.,Jermini,M.and R.B.(1995)Detection of non-culturable Vibrio cholerae O139,by Luthy,J.(1991)Detection of Escherichia coli and identi?cation of
PCR and?uorescent antibody methods,in laboratory micro-
?1997The Society for Applied Bacteriology,Journal of Applied Microbiology83,727–736
PCR DETECTION OF FOODBORNE PATHOGENS735 cosms.World Journal of Microbiology and Biotechnology11,597–Rosenberg,H.F.,Ackerman,S.J.and Tenen,D.G.(1990)An alter-598.
native method for labeling oligonucleotide probes for screening Jinneman,K.C.,Trost,P.A.,Hill,W.E.et al.(1995)Comparison cDNA libraries.BioTechniques8,384.
of template preparation methods from foods for ampli?cation of
Sethabutr,O.,Venkatesan,M.,Murphy,G.S.,Eampokalap,B., Escherichia coli O157Shiga-like toxins type I and II DNA by Hoge,C.W.and Echeverria,P.(1993)Detection of Shigellae and multiplex polymerase chain reaction.Journal of Food Protection
enteroinvasive Escherichia coli by ampli?cation of the invasion 58,722–726.plasmid antigen H DNA sequence in patients with dysentery. Koch,W.H.,Payne,W.L.,Wentz,B.A.and Cebula,T.A.(1993)
Journal of Infectious Diseases167,458–461.
Rapid polymerase chain reaction method for the detection of Shangkuan,Y.H.,Show,Y.S.and Wang,T.M.(1995)Multiplex Vibrio cholerae in foods.Applied and Environmental Microbiology
polymerase chain reaction to detect toxigenic Vibrio cholerae and 59,556–560.to biotype Vibrio cholerae O1.Journal of Applied Bacteriology79, Kwaga,J.,Iversen,J.O.and Misra,V.(1992)Detection of patho-
264–273.
genic Yersinia enterocolitica by polymerase chain reaction and Simon,M.C.,Gray,D.I.and Cook,H.(1996)DNA extraction and digoxigenin-labeled polynucleotide probes.Journal of Clinical
PCR methods for the detection of Listeria monocytogenes in cold Microbiology30,2668–2673.smoked salmon.Applied and Environmental Microbiology62,822–
824.
Lampel,K.A.,Jagow,J.A.,Trucksess,M.and Hill,W.E.(1990)
Polymerase chain reaction for detection of invasive Shigella?ex-Slavik,M.F.,Wang,R.-F.and Cao,W.-W.(1993)Development neri in food.Applied and Environmental Microbiology56,1536–
and evaluation of PCR method for diagnosis of Mycoplasma gal-1540.lisepticum infection in chicken.Molecular and Cellular Probes7, Lantz,P.G.,Hahnhagerdal,B.and Radstrom,P.(1994)Sample
459–463.
preparation methods in PCR-based detection of food pathogens.Song,J.H.,Cho,H.,Park,M.Y.,Na,D.S.,Moon,H.B.and Pai, Trends in Food Science and Technology5,384–389.
C.H.(1993)Detection of Salmonella typhi in the blood of patients Lawrence,L.M.and Gilmour,A.(1994)Incidence of Listeria spp.with Typhoid fever by polymerase chain reaction.Journal of and Listeria monocytogenes in a poultry processing environment
Clinical Microbiology31,1439–1443.
and in poultry products and their rapid con?rmation by multiplex Stone,G.G.,Oberst,R.D.,Hays,M.P.,McVey,S.and Chengappa, PCR.Applied and Environmental Microbiology60,4600–4604.
M.M.(1994)Detection of Salmonella serovars from clinical sam-Lee,C.Y.,Pan,S.F.and Chen,C.H.(1995)Sequence of a cloned ples by enrichment broth cultivation-PCR procedure.Journal of PR72H fragment and its use for detection of Vibrio para-
Clinical Microbiology32,1742–1749.
haemolyticus in shell?sh with the PCR.Applied and Environmental Strachan,N.J.C.and Gray,D.I.(1995)A rapid general method for Microbiology61,1311–1317.
the identi?cation of PCR products using a?ber-optic biosensor Makino,S.I.,Okada,Y.and Maruyama,T.(1995)A new method and its application to the detection of Listeria.Letters in Applied for direct detection of Listeria monocytogenes from foods by PCR.
Microbiology21,5–9.
Applied and Environmental Microbiology61,3745–3747.Tada,J.,Ohashi,T.,Nishimura,N.et al.(1992)Detection of the Muir,P.,Nicholson,F.,Jhetam,M.,Neogi,S.and Banatvala,J.E.
thermostable direct hemolysin gene(tdh)and the thermostable (1993)Rapid diagnosis of enterovirus infection by magnetic bead direct hemolysin-related hemolysin gene(trh)of Vibrio para-extraction and polymerase chain reaction detection of enterovirus
haemolyticus by polymerase chain reaction.Molecular and Cellular RNA in clinical specimens.Journal of Clinical Microbiology31,Probes6,477–487.
31–38.
Thomas,E.G.,King,R.K.,Burchak,J.and Gannon,V.P.J.(1991) Nakajima,H.,Inoue,M.,Mori,T.,Itoh,K.-I.,Arakawa,E.and Sensitive and speci?c detection of L.monocytogenes in milk and Watanabe,H.(1992)Detection and identi?cation of Yersinia
ground beef with PCR.Applied and Environmental Microbiology pseudotuberculosis and pathogenic Yersinia enterocolitica by an57,2576–2580.
improved polymerase chain reaction method.Journal of Clinical
Tsai,Y.L.,Palmer,C.J.and Sangermano,L.R.(1993)Detection of Microbiology30,2484–2486.Escherichia coli in sewage and sludge by polymerase chain reaction. Ra?i,F.,Holland,M.A.,Hill,W.E.and Cerniglia,C.E.(1995)
Applied and Environmental Microbiology59,353–357.
Survival of Shigella?exneri on vegetables and detection by poly-Victor,T.,du Toit,R.,van Zyl,J.,Bester,A.J.and van Helden,
P.D.(1991)Improved method for the routine identi?cation of merase chain reaction.Journal of Food Protection58,727–732.
Rahn.,K.,De Grandis,S.A.,Clarke,R.C.et al.(1992)Ampli?cation toxigenic Escherichia coli by DNA ampli?cation of a conserved of an invA gene sequence of Salmonella typhimurium by poly-
region of the heat-labile toxin A subunit.Journal of Clinical merase chain reaction as a speci?c method of detection of Salmon-Microbiology29,158–61.
ella.Molecular and Cellular Probes6,271–279.
Wang,R.-F.,Beasley,J.N.,Cao,W.-W.,Slavik,M.F.and Johnson, Rasmussen,H.N.,Rasmussen,O.F.,Christensen,H.and Olsen,M.G.(1993b)Development of PCR method speci?c for Marek’s J.E.(1995)Detection of Yersinia enterocolitica O-3in faecal sam-
disease virus.Molecular and Cellular Probes7,127–131.
ples and tonsil swabs from pigs using IMS and PCR.Journal of Wang,R.-F.,Campbell,W.,Cao,W.-W.,Ward,C.,Steele,R.S. Applied Bacteriology78,563–568.
and Cerniglia,C.E.(1996c)Detection of Pasteurella pneumotropica Read,S.C.,Clarke,R.C.,Martin,A.et al.(1992)Polymerase chain in laboratory mice and rats by https://www.wendangku.net/doc/df729351.html,boratory Animal Science reaction for detection of verocytotoxigenic Escherichia coli isolated
46,81–85.
from animal and food sources.Molecular and Cellular Probes6,Wang,R.-F.,Cao,W.-W.,Campbell,W.L.,Hairston,L.,Franklin, 153–161.
W.and Cerniglia,C.E.(1994b)The use of PCR to monitor the ?1997The Society for Applied Bacteriology,Journal of Applied Microbiology83,727–736
736R.-F.WANG ET AL.
population abundance of six human intestinal bacterial species in method for direct detection of low numbers of Campylobacter
jejuni.Journal of Rapid Methods and Automation in Microbiology an in vitro semicontinuous culture system.FEMS Microbiology
1,101–108.
Letters124,229–238.
Wernars,K.,Delfgou,E.,Soentoro,P.S.and Notermans,S.(1991) Wang,R.-F.,Cao,W.-W.and Cerniglia,C.E.(1996a)PCR detec-
Successful approach for the detection of low numbers of entero-tion and quantitation of predominate anaerobic bacteria in human
toxigenic Escherichia coli in minced meat by using the polymerase and animal fecal samples.Applied and Environmental Microbiology
chain reaction.Applied and Environmental Microbiology57,1914–62,1242–1247.
1919.
Wang,R.-F.,Cao,W.-W.,Franklin,W.,Campbell,W.L.and Cer-
Wernars,K.,Heuvelman,C.J.,Chakraborty,T.and Notermans, niglia,C.E.(1994a)A16S rDNA-based PCR method for rapid
S.H.W.(1991)Use of the PCR for direct detection of Listeria and speci?c detection of Clostridium perfringens in food.Molecular
monocytogenes in soft cheese.Journal of Applied Bacteriology70, and Cellular Probes8,131–138.
121–126.
Wang,R.-F.,Cao,W.-W.and Johnson,M.G.(1991)Development
Widjojoatmodjo,M.N.,Fluit,A.C.,Torensma,R.,Keller,B.H.I. of a16S rRNA-based oligomer probe speci?c for Listeria mono-
and Vechoef,J.(1991)Evaluation of the magnetic immuno PCR cytogenes.Applied and Environmental Microbiology57,3666–3670.
assay for rapid detection of Salmonella.European Journal of Clini-Wang,R.-F.,Cao,W.-W.,Johnson,M.G.and Cerniglia,C.E.
cal Microbiology10,935–938.
(1995)Comparison of PCR cycler machines for rapid and sensitive
Wiedmann,M.,Stolle,A.and Batt,C.A.(1995)Detection of Listeria detection of pathogens.The Rapid Cyclist3,12–14.monocytogenes in surface swabs using a non-radioactive poly-Wang,R.-F.,Cao,W.-W.and Johnson.,M.G.(1992a)16S rRNA-merase chain reaction-coupled ligase chain reaction assay.Food based probes and polymerase chain reaction method to detect Microbiology12,151–157.
Listeria monocytogenes cells added to foods.Applied and Environ-Wilson,I.G.,Cooper,J.E.and Gilmour,A.(1991)Detection of mental Microbiology58,2827–2831.enterotoxigenic Staphylococcus aureus in dried skimmed milk:use Wang,R.-F.,Cao,W.-W.,Wang,H.and Johnson,M.G.(1993a)A of the polymerase chain reaction for ampli?cation and detection 16S rRNA-based DNA probe and PCR method speci?c for Lis-of staphylococcal enterotoxin genes entB and entC1and the ther-teria ivanovii.FEMS Microbiology Letters106,85–92.monuclease gene nuc.Applied and Environmental Microbiology57, Wang,R.-F.,Luneau,A.,Cao,W.-W.and Cerniglia,C.E.(1996b)1793–1798.
PCR detection of polycyclic aromatic hydrocarbons degrading Yang,T.H.and Chen,T.R.(1993)Use of the polymerase chain Mycobacteria in soil.Environmental Science and Technology30,reaction for speci?c detection of type A,D,and E enterotoxigenic 307–311.Staphylococcus aureus in foods.Applied Microbiology and Biotech-
nology37,685–690.
Wang,R.-F.,Slavik,M.F.and Cao,W.-W.(1992b)A rapid PCR
?1997The Society for Applied Bacteriology,Journal of Applied Microbiology83,727–736