PCR-ELISA de ec ion o Esche ichia coli in milk
P. Daly, T. Collie and S. Doyle
Depa men o Biology, Na ional Uni e si y o I eland, Maynoo h, I eland
2001/232: ecei ed 6 Augus 2001 and accep ed 19 Decembe 2001
P. DALY, T. COLLIER AND S. D O YLE. 2002.
Aims: The pu pose o his s udy was o de elop a eliable molecula p ocedu e o he
de ec ion o Esche ichia coli in milk.
Me hods and Resul s: Robus and expedi ious DNA ex ac ion and PCR echniques we e
e alua ed using Enzyme-Linked Immunoso ben Assay (ELISA) de ec ion o bio in-labelled
amplicons o acili a e op imal de ec ion o E. coli DNA.
Conclusions: I was ound ha 5 E. coli colony- o ming uni s (c u) could be de ec ed pe PCR
eac ion using he PCR-ELISA sys em, equa ing o a sensi i i y o de ec ion o 100 E. coli
c u ml
)1
pas eu ized milk.
Signi icance and Impac o he S udy: This app oach should acili a e e alua ion o milk
con amina ion and enable apid de ec ion o E. coli mas i is, leading o co ec deploymen o
ele an an ibio ic he apy and imp o ed animal wel a e.
INTRODUCTION
In ecen yea s Esche ichia coli has become ecognized as a
se ious ood-bo ne pa hogen and has been associa ed wi h
nume ous ou b eaks o disease in he UK, Japan and USA
(Uy endaele e al. 1999; Sco e e al. 2000). Raw milk and
dai y p oduc s, such as pas eu ized milk and yoghu
con amina ed wi h E. coli, ha e been he main cause o
se e al ou b eaks o milk-bo ne disease since he 1980s and
hus emain a se ious heal h isk (Allmann e al. 1995; Seo
e al. 1998). De ec ion and enume a ion o E. coli in ood
p oduc s can be assessed by a ious me hods including pla e
coun ing, immuno-magne ic sepa a ion, low cy ome y and
ch omogenic- luo ogenic subs a e echnology (Venka es-
wa an e al. 1996; de Boe 1998; Seo e al. 1998; Yu 1998).
The s anda d de ec ion me hod o E. coli in ol es he use
o subs a es ha de ec E. coli and can be ime-consuming.
Up o 97% o E. coli p oduce b-
D
-glucu onidase which
hyd olyses 4-me hylumbelli e yl-b-
D
-glucu onide (MUG)
o o m a blue luo escen p oduc (Venka eswa an e al.
1996; Mana i 2000). Howe e , a mu a ion in he uidA gene
in E. coli leads o alse-nega i e iden i ica ion when using
b-
D
-glucu onidase as a me hod o de ec ion, while ce ain
s ains o E. coli, such as O157, ail o p oduce his enzyme
(Yokoigawa e al. 1999). In addi ion, b-
D
-glucu onidase is
also p oduced by membe s o he Salmonella,Shigella and
some Ye sinia spp., while S aphylococcus spp. can also
hyd olyse MUG. Thus, mo e accu a e me hods o de ec -
ing E. coli a e necessa y (de Boe 1998).
Polyme ase chain eac ion (PCR) has led o apid and
sensi i e de ec ion o E. coli om clinical samples
and a ious ood ypes (F a amico e al. 2000; McKillip
and D ake 2000; Hsu and Tsen 2001). Howe e , many ood
ypes con ain PCR inhibi o s which co-pu i y wi h he
a ge DNA (Gonza
´lez e al. 1999), he eby equi ing
ex ensi e sample p epa a ion o emo e, dilu e o inac i a e
inhibi o s p io o PCR ampli ica ion (F a amico e al.
2000). In many cases, he low le el o E. coli in ood ypes
equi es a p e-en ichmen s ep o o e come poo sensi i i y,
which inc eases he o e all assay ime by up o 24 h (Seo
e al. 1998; Sco e e al. 2000). Thus, a mo e sensi i e,
speci ic and apid me hod o de ec ing E. coli is equi ed.
Alanine acemase is p oduced by all E. coli s ains,
including O157, and is essen ial o cell-wall biosyn hesis by
p o iding
D
-alanine o pep idoglycan. Thus, de ec ion o
he alanine acemase (al ) gene may be use ul o he
de ec ion o E. coli in oods (Yokoigawa e al. 1999). In he
p esen s udy, he success ul design and applica ion o a
obus PCR-ELISA o he de ec ion o low le els E. coli in
milk is desc ibed.
Co espondence o: D S. Doyle, Depa men o Biology, Na ional Uni e si y o
I eland, Maynoo h, Co. Kilda e, I eland (e-mail: [email p o ec ed]).
ª2002 The Socie y o Applied Mic obiology
Le e s in Applied Mic obiology 2002, 34, 222–226
MATERIALS AND METHODS
Bac e ial s ain and a i icial specimen p epa a ion
Esche ichia coli s ain JM109 was ob ained om P omega
(Madison, WI, USA). Esche ichia coli-adul e a ed milk
specimens we e p epa ed as ollows. An E. coli cul u e was
g own in Lu ia-Be ani (LB) B o h a 37°C o 16 h wi h
shaking. A 25 ml aliquo o he cul u e was cen i uged a
3200 g o 15 min. A e emo al o he supe na an luid,
he pelle was esuspended in 1 ml esh pas eu ized milk,
aw milk o phospha e-bu e ed saline (0Æ05% ( / ) Tween-
20; PBST) and each sample se ially dilu ed in unadul e a ed
ma ix o de e mine he sensi i i y o PCR ampli ica ion. A
100 ll aliquo o each dilu ion was also pla ed, in iplica e,
on LB Aga and colony coun ing was pe o med using an
Eagle-Eye II gel documen a ion sys em (S a agene, La
Jolla, CA, USA). F esh pas eu ized milk and aw milk we e
indi idually pla ed o de e mine he p esence o na u ally-
con amina ing mic o lo a.
DNA ex ac ion
Milk samples (100 ll) con aining E. coli (0–10
4
c u ml
)1
)
we e each mixed wi h 1 ml PBST, o exed, and cen i-
uged a 10 000 g o 5 min. This p ocedu e was pe o med
o pelle he bac e ial cells and o emo e in e e ing
amoun s o p o ein and lipid in milk samples, which may
in e e e wi h PCR ampli ica ion. Following emo al o he
supe na an luid, DNA was ex ac ed using he ollowing
p ocedu es.
Alkaline ex ac ion. The alkaline ex ac ion p ocedu e was
a modi ica ion o he me hod p oposed by Milla e al.
(2000). The bac e ial pelle was esuspended in 1 ml alka-
line ex ac ion solu ion (0Æ5moll
)1
sodium hyd oxide,
0Æ05 mol l
)1
sodium ci a e). This mix u e was shaken o
10 min and cen i uged a 13 000 g o 5 min. The supe -
na an luid was again emo ed and he pelle esuspended
in 500 ll0Æ5 mol l
)1
T is-HCl pH 8Æ0, ollowed by cen-
i uga ion a 13 000 g o 5 min. This s ep was epea ed.
The inal pelle was esuspended in 100 ll 10 mmol l
)1
T is-HCl, 1 mmol l
)1
EDTA pH 8Æ0, and placed in a
hea ing block o 1 h a 100°C. The sample was hen eeze–
hawed wice, cen i uged a 13 000 g o 15 min and he
supe na an luid emo ed o PCR analysis.
De e gen ex ac ion. The ex ac ion me hod o Yokoig-
awa e al. (1999) o de ec E. coli s ains in oods, including
aw milk, was employed. He e, ollowing he ini ial
cen i uga ion o milk samples in PBST, he supe na an
luid was emo ed and 100 ll lysis bu e (10% (w/ ) NaCl,
2% ( / ) Tween-20 and 2 mmol l
)1
EDTA) was added.
Samples we e hen ea ed a 100°C on a hea ing block o
10 min and cen i uged a 5000 g o 5 min. Supe na an
luids we e hen used di ec ly as PCR empla es.
Sodium iodide (NaI) ex ac ion. The NaI ex ac ion
p ocedu e was ha p oposed by Ishizawa e al. (1991) o
isola ion o DNA om se um.
Oligonucleo ide p ime s and DNA ampli ica ion
P ime s speci ic o a conse ed egion si ua ed wi hin he
E. coli alanine acemase gene we e selec ed (Yokoigawa e al.
1999) using a modi ied ampli ica ion p ocedu e: o wa d: 5¢
Bio in-CTGGAAGAGGCTAGCCTGGACGAG 3¢and
e e se: 5¢-AAAATCGGCACCGGTGGAGCGATC-3¢
(Sigma-Genosys, Camb idge, UK). A 5 ll olume o each
ex ac ed sample was used o PCR (PCR Exp ess
The mal Cycle ; Hybaid, Ash o d, Middlesex, UK), which
also con ained 10 mmol l
)1
T is-HCl pH 9Æ0, 50 mmol l
)1
KCl, 0Æ1% ( / ) T i on X-100, 2Æ0 mmol l
)1
MgCl
2
,
200 lmol l
)1
o each dNTP (P omega), 1 mol l
)1
be aine
(Sigma) and 1Æ0lmol l
)1
o he abo e p ime s in a o al
olume o 49Æ5ll. Ho -s a PCR was pe o med on each
sample by adding 1Æ25 U Taq polyme ase (P omega) a e
he ini ial 6 min dena u a ion s ep a 95°C. The PCR
eac ion was hen con inued wi h 35 cycles consis ing o 20 s
dena u a ion a 95°C, p ime annealing/ex ension a 72°C
o 90 s, and a inal ex ension o 5 min a 72°C. A 10 ll
aliquo o each PCR p oduc was subjec ed o 1% (w/ )
aga ose gel elec opho esis con aining 0Æ5lgml
)1
e hidium
b omide (Sigma) o 30 min a 100 V. Amplicon isualiza-
ion (366 bp) was pe o med using an Eagle-Eye II gel
documen a ion sys em (S a agene).
Mic opla e p epa a ion and ELISA o ma
S ep a idin-coa ed mic opla es we e p epa ed by coa ing
mic owells (Nunc Maxiso p, Roskilde, Denma k) wi h
s ep a idin (2Æ5lgml
)1
) in 50 mmol l
)1
sodium ca bona e
pH 9Æ4, and s abilized by he addi ion o 1% (w/ ) albumin
in he same diluen . Bio inyla ed PCR p oduc s we e dilu ed
1/20 in 6·SSC o gi e a inal olume o 200 ll; hey we e
hen added o s ep a idin-coa ed mic owells and incuba ed
a 37°C o 30 min. A e wo washes wi h PBST, 100 llo
125 mmol l
)1
NaOH, 100 mmol l
)1
NaCl we e added o he
mic owells, incuba ed a oom empe a u e o 3 min and
washed ou imes wi h PBST. A 100 ll olume o
dini ophenol (DNP)-labelled oligonucleo ide (100 ng ml
)1
;
5¢(DNP)
3
-ATATTCACCGGCTGACGAAC-3¢)in6·
SSC/0Æ1% (w/ ) sodium dodecyl sulpha e was added o
each mic owell and incuba ed o 1 h a 60°C, ollowed by
washing ou imes wi h PBST. Mic owells we e hen
blocked wi h 2Æ5% (w/ ) milk powde in PBST a 20°C o
1 h. Following blocking solu ion emo al, IgG [an i-DNP]
E. COLI PCR-ELISA 223
ª2002 The Socie y o Applied Mic obiology, Le e s in Applied Mic obiology,34, 222–226
ho se adish pe oxidase conjuga e was added, incuba ed a
20°C o 30 min and washed ou imes wi h PBST.
Subs a e (100 ll e ame hylbenzidine) was hen added and
incuba ed a 20°C o 15 min. The eac ion was e mina ed
by he addi ion o 0Æ5 mol l
)1
sulphu ic acid and measu ed
spec opho ome ically a 450/630 nm (Dyna ech MRX
Dynex Technologies, Ash o d, Middlesex, UK).
RESULTS
Ex ac ion o E. coli and de ec ion o al gene
amplicons by aga ose gel elec opho esis
Esche ichia coli was cul u ed in LB b o h and added o aw
milk, esh pas eu ized milk and PBST. Following pla ing o
each dilu ion on LB aga , he numbe s o colony- o ming
uni s pe millili e (c u ml
)1
) we e de e mined. Esche ichia
coli DNA was ex ac ed om adul e a ed samples anging
om 10
0
o 10
4
c u ml
)1
using he alkaline, de e gen and
NaI ex ac ion p ocedu es.
No E. coli DNA was de ec able in ei he unadul e a ed
pas eu ized o aw milk, he la e ha ing been ob ained
unde s e ile condi ions. Bac e ial DNA was success ully
ex ac ed om adul e a ed aw and pas eu ized milk sam-
ples, and PBST, by he alkaline ex ac ion and NaI me hods.
A minimum o 50 E. coli c u we e ep oducibly de ec able
by PCR using he al gene oligonucleo ide p ime s (Table 1
and Fig. 1). Since 5 ll ex ac ed sample (ini ial olume
100 ll) we e used in each PCR eac ion, he PCR de ec ion
limi was de e mined o be 10
3
c u ml
)1
. Signi ican ly, he
de e gen ex ac ion me hod p oposed by Yokoigawa e al.
(1999) ailed o esul in he gene a ion o any PCR p oduc
(Table 1). In ac , ampli ica ion o a ge DNA ailed e en
a e high le els o E. coli (10
8
c u ml
)1
) we e ex ac ed by
he de e gen me hod (da a no shown). I was also ound
Table 1 PCR ampli ica ion o Esche ichia coli DNA ollowing ex ac ion by sodium iodide, de e gen and hea om aw milk (milk (R), pas eu ized
milk (milk (P) and PBST (con ol diluen ). The in ensi y o e hidium b omide amplicon binding is gi en by he ollowing abb e ia ions: high (3 +),
mode a e (2 +), low (+) and nega i e ())
Ex ac ion me hods
E. coli (c u 5 ll
)1
Sodium iodide De e gen Hea
PCR empla e) Milk (R) Milk (P) PBST Milk (R) Milk (P) PBST Milk (R) Milk (P) PBST
0)))))))))
0Æ5)))))))))
5))))))))+
50 + 2 + 2 + )))))2+
500 3+ 3+ 3+ )))))3+
Fig. 1 Aga ose gel elec opho esis o 366 bp PCR p oduc s om alkaline ex ac ion o aw milk, pas eu ized milk and PBST con aining 0–500 c u
Esche ichia coli. Lane 1: nega i e con ol; lanes 2–4: aw milk, pas eu ized milk and PBST, ollowing alkaline ex ac ion, con aining no E. coli; lanes 5–
7: aw milk, pas eu ized milk and PBST, ollowing alkaline ex ac ion, con aining 5 c u E. coli; lanes 8–10: aw milk, pas eu ized milk and PBST,
ollowing alkaline ex ac ion, con aining 50 c u E. coli; lanes 11–13: aw milk, pas eu ized milk and PBST, ollowing alkaline ex ac ion, con aining
500 c u E. coli; lane 14: pu i ied E. coli DNA (s ain JM109); lane 15: 1 kbp molecula size ma ke
224 P. DALY ET AL.
ª2002 The Socie y o Applied Mic obiology, Le e s in Applied Mic obiology,34, 222–226
ha hea ea men alone o bac e ial cells (100°C o
15 min) ob ained om ei he aw o pas eu ized milk
was insu icien o acili a e success ul PCR ampli ica ion o
E. coli DNA, ye DNA was de ec able a e hea ea men
only, a iden ical E. coli c u ml
)1
, in PBST (Table 1).
Aga ose gel elec opho esis o PCR p oduc s shows he
de ec ion o 50 and 500 c u using he alkaline ex ac ion
me hod o DNA ex ac ion om PBST and aw milk,
espec i ely (Fig. 1). Occasionally, as ew as 5 E. coli c u
(equi alen o 100 c u ml
)1
) we e de ec able a e alkaline
ex ac ion om pas eu ized milk. Howe e , e hidium b o-
mide s aining could no ep oducibly de ec E. coli a his
low le el.
PCR-ELISA o al gene amplicons
Following amplicon de ec ion by gel elec opho esis, PCR
p oduc s we e hen u he analysed by ELISA in an a emp
o imp o e he sensi i i y o de ec ion. The esul s o he
ELISA o PCR p oduc s om alkaline ex ac ion a e p esen-
ed in Fig. 2. Resul s show speci ic de ec ion o 50–500 c u by
he DNP-labelled oligonucleo ide p obe when E. coli was
ex ac ed om aw milk and PBST. Pas eu ized milk sam-
ples showed enhanced ex ac ion o E. coli DNA ela i e o
aw milk and PBST, whe eby as ew as 5 c u could be eliably
de ec ed using PCR-ELISA a e DNA pu i ica ion by he
alkaline ex ac ion me hod; his con i med he inc eased
sensi i i y o he ELISA de ec ion me hod o e con en ional
aga ose gel elec opho esis and e hidium b omide de ec ion
o DNA (i.e. 10
2
s 10
3
c u ml
)1
, espec i ely).
DISCUSSION
A apid and sensi i e PCR-ELISA sys em o de ec ing
E. coli in milk has been de eloped. Using se ial dilu ions o
E. coli in aw and pas eu ized milk and PBST, he speci ic
de ec ion o low le els o E. coli and he emo al o po en ial
inhibi o y subs ances by alkaline ex ac ion o NaI ex ac-
ion ha e been demons a ed. The PCR-ELISA used in his
s udy de ec ed be ween 5 and 50 E. coli c u and did no
equi e a p e-en ichmen s ep.
Hsu and Tsen (2001) ound le els o 10
6
c u ml
)1
(10
3
E.
coli c u ml
)1
using pla e coun aga ). Bee ens e al. (2000)
obse ed a mean le el o 119 E. coli c u ml
)1
in 58 samples
o aw milk. The le el o endogenous mic o lo a disco e ed
in aw milk in he p esen s udy was 2 ·10
2
c u ml
)1
, wi h
obse ed le els o E. coli be ween 1 and 10 c u ml
)1
. This
lowe le el o bac e ia in aw milk could be a ibu ed o
ob aining he milk om cows unde asep ic condi ions and
om heal hy cows wi h no e idence o mamma y in ec ion.
Uy endaele e al. (1999) epo ed ha he p esence o
10
6
c u ml
)1
o endogenous mic o lo a did no in luence he
de ec ion limi o hei PCR assay, which was in ag eemen
wi h ou obse a ions. I should be no ed ha he al
p ime s used in his s udy de ec a wide ange o E. coli
s ains bu also lead o he de ec ion o Shigella spp.
(Yokoigawa e al. 1999). Howe e , he incidence le els o
Shigella in oods is o dina ily e y low (Gonza
´lez e al. 1999)
and he e o e would no be expec ed o complica e he
de ec ion limi s o he PCR-ELISA assay p esen ed he e.
Yokoigawa e al. (1999) quo e a sensi i i y o de ec ion o
10
3
E. coli cells ml
)1
using de e gen ex ac ion and an
iden ical p ime pai o ha employed in he p esen s udy.
Howe e , i p o ed impossible o eplica e his inding,
possibly due o he inhibi o y na u e o EDTA in he PCR
eac ion. In o de o ob ain an equi alen le el o sensi i i y
o ha o Yokoigawa e al., he ollowing modi ica ions
we e equi ed. Fi s , an inc ease in PCR cycle numbe
om 25 o 35 cycles had a signi ican e ec , esul ing in a
leas a 10
6
inc ease in sensi i i y o de ec ion. Secondly, he
Fig. 2 PCR-ELISA analysis o aw milk (j),
pas eu ized milk ( ) and PBST (h) con-
aining 0–500 c u Esche ichia coli ob ained
ollowing alkaline ex ac ion p ocedu e. Neg-
a i e and posi i e con ols a e also indica ed.
Assay cu -o (ho izon al line) was calcula ed
by eplica e analysis o eigh nega i e speci-
mens and was de e mined o be 0Æ105 O.D.
uni s (mean
nega i es
+ 2 s anda d de ia ions,
i.e. 0Æ065 + 2 (0Æ02))
E. COLI PCR-ELISA 225
ª2002 The Socie y o Applied Mic obiology, Le e s in Applied Mic obiology,34, 222–226
wash/cen i uga ion s ep p io o ex ac ion was included o
enhance he eco e y o bac e ia and also o help emo e he
high le els o a and p o ein. In e es ingly, he elimina ion
o po en ial PCR inhibi o s by he inclusion o a washing
s ep while eco e ing he bac e ial pelle p io o DNA
ex ac ion was also achie ed by Uy endaele e al. (1999).
Thi dly, ho -s a PCR was used o p e en educed ac i i y
o Taq polyme ase du ing he ini ial dena u a ion s ep.
Finally, be aine (1 mol l
)1
) was included as a PCR addi i e
o enhance ampli ica ion. In es iga ion in o he ailu e o
ampli ica ion using he me hod o Yokoigawa e al. (1999)
illus a ed ha e en when 10
6
c u E. coli we e added o he
PCR mas e mix con aining 5 ll o de e gen ex ac ion
bu e , inhibi ion o ampli ica ion esul ed.
The highe sensi i i y o E. coli de ec ion in pas eu ized
milk samples using alkaline ex ac ion can be explained by
enhanced emo al o inhibi o s, he eby acili a ing
inc eased ampli ica ion e iciency. Fu he mo e, ailu e o
sample hea ing alone o elease de ec able E. coli DNA om
milk u he highligh s he impo ance o he emo al o
inhibi o y compounds p io o ampli ica ion.
The de elopmen o his no el PCR-ELISA me hod
o e s se e al ad an ages o e adi ional mic obiological
cul u e echniques. The PCR s ep akes jus o e 1 h o
comple e due o he simul aneous annealing and polyme -
iza ion a 72°C. Speci ic amplicon de ec ion ia he DNP
oligonucleo ide p obe used in he ELISA con e s inc eased
assay speci ici y wi h op imal sensi i i y. The me hod is
s aigh o wa d, does no equi e expensi e equipmen and
should be capable o de ec ing he sugges ed E. coli O157
in ec i e dose (4–24 o ganisms) as p oposed by S achan
e al. (2001). Au oma ion and mul iple sample analysis can
also be mo e easily pe o med on s ep a idin-coa ed
mic o i e pla es in compa ison wi h aga ose gel elec o-
pho esis. In addi ion, he subjec i i y o band iden i ica ion
and he use o ca cinogenic DNA binding agen s a e also
elimina ed. This no el PCR-ELISA should help in he
assessmen o he hygienic quali y o milk collec ed om
a ms and acili a e apid diagnosis o E. coli mas i is, which
would allow o ea ly an ibio ic he apy helping o educe
loss in milk p oduc ion and damage o mamma y issue.
ACKNOWLEDGEMENTS
The inancial assis ance o En e p ise I eland and T idel a
De elopmen Limi ed is g a e ully acknowledged.
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