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h ps://dx.doi.o g/10.1016/j.aca.2013.12.017
h p://hdl.handle.ne /10251/65319
Else ie
San iago Felipe, S.; To ajada-Gena o, LA.; Puchades, R.; Maquiei a Ca ala, Á. (2014).
Recombinase polyme ase and enzyme-linked immunoso ben assay as a DNA
ampli ica ion-de ec ion s a egy o ood analysis. Analy ica Chimica Ac a. 811:81-87.
doi:10.1016/j.aca.2013.12.017.
1
RECOMBINASE POLYMERASE AND ENZYME-LINKED
1
IMMUNOSORBENT ASSAY AS A DNA AMPLIFICATION-DETECTION
2
STRATEGY FOR FOOD ANALYSIS
3
4
S. San iago-Felipe, L.A. To ajada-Gena o, R. Puchades, A. Maquiei a
5
Cen o de Reconocimien o Molecula y Desa ollo Tecnológico (IDM) - Depa amen o de
6
Química, Uni e si a Poli ècnica de València, Camino de Ve a s/n, 46022 Valencia, Spain.
7
email: amaquiei [email p o ec ed] .es
8
9
10
ABSTRACT
11
Polyme ase chain eac ion in conjunc ion wi h enzyme-linked immunoso ben assay
12
(PCR-ELISA) is a well-es ablished echnique ha p o ides a sui able apid, sensi i e,
13
and selec i e me hod o a b oad ange o applica ions. Howe e , he need o p ecise
14
apid empe a u e cycling o PCR is an impo an d awback ha can be o e come by
15
employing iso he mal ampli ica ion eac ions such as ecombinase polyme ase
16
ampli ica ion (RPA). The RPA–ELISA combina ion is p oposed o ampli ica ion a a
17
low, cons an empe a u e (40 ºC) in a sho ime (40 min), o he hyb idisa ion o
18
labelled p oduc s o speci ic 5’-bio inyla ed p obes/s ep a idin in coa ed mic o i e
19
pla es a oom empe a u e, and o de ec ion by colo ime ic immunoassay. RPA-
20
ELISA was applied o sc een common sa e y h ea s in oods u s, such as alle gens
21
(hazelnu , peanu , soybean, oma o, and maize), gene ically modi ied o ganisms (P35S
22
and TNOS), pa hogenic bac e ia (Salmonella spp. and C onobac e spp.), and ungi
23
(Fusa ium spp.). Sa is ac o y sensi i i y and ep oducibili y esul s we e achie ed o
24
all he a ge s. The RPA-ELISA echnique does away wi h he mocycling and p o ides
25
a sui able sensi i e, speci ic, and cos -e ec i e me hod o ou ine applica ions, and
26
p o es pa icula ly use ul o esou ce-limi ed se ings.
27
28
Keywo ds: iso he mal ampli ica ion; ELISA; alle gen; GMO; pa hogen; ood
29
sa e y
30
31
2
1. In oduc ion
32
Analy ical me hods o as , eliable, sensi i e and cos -e ec i e de ec ion a e
33
highly demanded in many a eas, including ood sa e y. Me hods based on he de ec ion
34
o nucleic acids o e in e es ing bene i s because DNA molecules show cons an
35
concen a ions, s abili y, and be e ex ac ion yields (e en om p ocessed and hea -
36
ea ed samples) han p o eins [1-2]. Fu he mo e, he ampli ica ion by polyme ase
37
chain eac ion (PCR) ensu es he equi ed sensi i i y le els. Consequen ly in ecen
38
yea s, he PCR-based me hods, e.g. RT-PCR, digi al PCR o mic oa ay, a e he gold
39
s anda d o he analysis o nucleic acids [2-4].
40
The demand o sequence-speci ic app oaches ha do no equi e labo ious o
41
expensi e de ec ion echnologies has led o he hyphena ion o PCR wi h enzyme-linked
42
immunoso ben assay (PCR-ELISA) [5-7]. This combines he high selec i i y o DNA-
43
based me hods wi h ELISA sensi i i y. This in eg a ion in ol es he hyb idisa ion o
44
labelled ampli ica ion p oduc s o speci ic cap u ed p obes in each mic o i e well, as
45
well as hei immunode ec ion. Al hough sensi i i y can be imp o ed using luo ome ic
46
o chemiluminiscen subs a es, PCR-ELISA me hods no mally employ colo ime ic
47
de ec ion because be e ep oducibili y, cos -by-assay, and s abili y a e achie ed [6-8].
48
The e o e, colo ime ic PCR-ELISA is a me hod ha is capable o p ocessing up o 96
49
o 384 assays simul aneously, i is po en ially au oma able, and only equi es he basic
50
ins umen s p esen in any diagnos ic labo a o y.
51
The he mal PCR echnique has i s limi a ions, such as equi ing p ecise
52
empe a u e con ol and apid he mocycling s eps among he empe a u es o
53
dissocia ion (95 ºC), annealing (55-65 ºC) and elonga ion (70 ºC) [6]. Ye he use o
54
o he enzymes (o a combina ion o enzymes) o mimic DNA eplica ion in i o has
55
eme ged as a solu ion o con en ional PCR polyme ases [9-10]. A he momen , wo
56
iso he mal ampli ica ion eac ions in combina ion wi h ELISA ha e been desc ibed:
57
loop-media ed iso he mal ampli ica ion (LAMP-ELISA) [11] and helicase-dependen
58
ampli ica ion (HDA-ELISA) [12]. They do no equi e expensi e ampli ica ion
59
equipmen and ha e been seen o be e y lexible and capable o simul aneously
60
p ocessing up o se e al hund eds o samples in a ew hou s. Howe e , hese eac ions
61
a e pe o med a om oom empe a u e (60 – 65 ºC), equi e an ini ial dena u a ion
62
s ep a 95 ºC (HDA) o need complex p ime s design (LAMP).
63
An inno a i e iso he mal ampli ica ion called ecombinase polyme ase
64
ampli ica ion (RPA) o e s in e es ing ad an ages [13-14]. This echnique acili a es he
65
3
binding o oligonucleo ide p ime s o empla e DNA. P ime s a e elonga ed by a s and-
66
displacing DNA polyme ase, called Bsu polyme ase, while single-s anded DNA-
67
binding p o eins s abilise ampli ica ion eac ion in e media es. Compa ed o o he
68
ampli ica ion enzymes, Bsu polyme ase main ains simila ac i i y in inhibi ing
69
en i onmen s, equi es a sho e incuba ion ime, ope a es a lowe empe a u es, is easy
70
o use and he ampli ied p oduc s do no need pos -ampli ica ion ea men . The
71
in eg a ion o DNA ampli ica ion and he de ec ion s ep en ails adjus ing se e al c i ical
72
a iables. Fo ins ance, RPA bu e con ains Ca bowax 20M, a high-molecula -weigh
73
polye hylene glycol, as a c owding agen o in luence he ecombinase kine ics.
74
Howe e , i is known ha hese molecules may a ec he solubili y, mel ing
75
empe a u e o iscosi y o he eac ion mix, and hence he ampli ied p oduc [15].
76
In his s udy, he hyphena ion o he RPA and ELISA me hodologies (RPA-
77
ELISA) is de eloped o ood sa e y applica ions. The echnical implemen a ion o
78
in eg a ed sc eening me hods in ood indus y can as ly help o simpli y he p ocess
79
and o educe cos s, hus making analy ical p ocedu es iendlie [1]. He e he
80
simul aneous de ec ion o di e en common ood h ea s is p oposed. Hazelnu , peanu
81
and soybean ha e been selec ed as ep esen a i e examples o he alle gens included in
82
p io i y lis s [16]. Toma o and maize ha e also been included because he e is g owing
83
conce n abou hei alle genici y associa ed wi h hei cu en widesp ead use [17-18].
84
P omo e 35S and e mina o NOS a e widely used o sc eening gene ically modi ied
85
o ganisms (GMOs) since se en y- wo pe cen o GMOs con ains a leas one o hese
86
sequences [19-20]. Salmonella spp., C onobac e spp. a e equen ly de ec ed as being
87
esponsible o ood con amina ion. Acco ding o he Food and Ag icul u e
88
O ganiza ion o he Uni ed Na ions and he Wo ld Heal h O ganiza ion, hey a e
89
conside ed o be included among he mos ele an pa hogens and hei absence is
90
equi ed in ood sa e y analysis [21-22]. Finally, Fusa ium spp. is one o mos equen
91
ungi ound in ood and eed de i a es, and i p oduces myco oxins ha cause se ious
92
heal h p oblems in bo h humans and li es ock [23-24].
93
94
4
2. Expe imen al
95
2.1 Ta ge genes
96
The selec ed genes o hazelnu , peanu , soybean, oma o, maize, GMO p omo e -
97
P35S, GMO e mina o -TNOS, Salmonella spp., C onobac e spp., and Fusa ium spp.
98
a e shown in Table 1. All he p ime s and p obes we e success ully checked o ele an
99
homologies by a BLASTN sea ch (h p://blas .ncbi.nlm.nih.go /).
100
101
2.2 Bac e ial and ungal s ains, oods u and DNA ex ac ion
102
Salmonella yphimu ium g oup B (CECT 443) and C onobac e sakazakii (ATCC
103
BBA-894) we e used as e e ence s ains. They we e isola ed and p o ided by he
104
GAIKER Technology Cen e (Bizkaia, Spain). Viable samples we e ob ained by
105
o e nigh cul u e on nu ien aga pla es (0.5% Pep one, 0.3% bee ex ac , 1.5% aga ,
106
0.5% NaCl, pH 7) a 37 ºC. Bac e ial inocula ion assays we e p epa ed by adding 10-
107
old se ial dilu ions o an 18-hou cul u e o each pa hogen in s e ile saline solu ion
108
(0.8% NaCl), co e ing a ange om 0 o 4104 CFU mL-1. Fusa ium monili o me
109
(CECT 2982) was used as he e e ence ungal s ain. I was isola ed and p o ided by
110
he Ins i u o Ag o o es al Medi e áneo (IAM), Uni e si a Poli ècnica de València
111
(UPV). Viable samples we e ob ained by cul u ing 4 days on nu ien aga pla es a
112
25ºC. Fungi inocula ion assays we e p epa ed by adding ungal mycelium (103-105 µg
113
o mycelium pe g o ood) om a 4-day cul u e. The ce i ied e e ence ma e ials
114
(CRM) con aining 0.05% o ansgenic B 11 maize (ERM-BF412 ) and 0.01% o
115
ansgenic RRS soybean (ERM-BF410gk) we e pu chased om he Ins i u e o
116
Re e ence Ma e ial and Measu emen s (Geel, Belgium). Food p oduc s we e bough in
117
local s o es. Genomic DNA was ex ac ed om bac e ial cul u es, ungal mycelium, and
118
ood samples using he DNeasy Blood & Tissue Ki (Qiagen, Inc., CA).
119
Inocula ion assays we e assessed a e aking in o accoun hei common
120
concen a ion in con amina ed oods (e.g., Salmonella spp. >102 CFU/mL) [28].
121
122
2.3 RPA-ELISA
123
RPA assays we e ca ied ou in a o al olume o 25 μL using he Twis Amp
124
Basic ki (Twis DX, Camb idge, UK). Reac ions con ained 480 nM o each 5’-
125
digoxigenin labelled p ime (Table 1), 15 ng o genomic DNA, 14 mM o Mg ace a e,
126
and 1× ehyd a ion bu e . Fi s ly, all he eagen s excep o he DNA empla e and Mg
127

5
ace a e we e p epa ed in a mas e mix, which was dis ibu ed in o each 0.2 mL eac ion
128
ube con aining he enzyme and he nucleo ides in a d ied pelle . Then, DNA was added
129
in o he ubes, and Mg ace a e was dispensed las ly. Since he RPA eac ion s a s as
130
soon as magnesium is added, he ubes we e immedia ely placed in o a hea ing o en
131
(Memme , model UF30, Ge many) a 40 ºC o 40 min.
132
Ampli ica ion p oduc s we e analysed in 96-well mic o i e ELISA pla es
133
(Co ning, USA). Fo his pu pose, 100 µL o s ep a idin (0.2 mg L-1) and bio inyla ed
134
p obes (20 nM), dilu ed in coa ing bu e (50 mM ca bona e bu e , pH 9.6) and we e
135
incuba ed o e nigh a 4 ºC. Double labelled oligonucleo ide (5’-bio in and 3’-
136
digoxigenin) was used as he posi i e con ol and non- a ge bio inyla ed
137
oligonucleo ide was he nega i e con ol (no complemen a y o any a ge ). Mic o i e
138
pla es we e washed 3 imes wi h PBS-T (phospha e-bu e ed saline con aining 0.05%
139
( / ) ween 20, pH 7.4) plus deionised wa e , and we e s o ed a 4 ºC o become s able
140
a less 1 mon h. Ampli ied p oduc s (1 L) we e mixed wi h 99 L o 5 hyb idisa ion
141
bu e (SSC, 1 saline sodium ci a e: NaCl 150 mM, sodium ci a e 15 mM, pH 7) and
142
hea ed a 95 ºC o 5 min o dena u e in o single s ains. Then dena u ed p oduc s (100
143
µL) we e dispensed in o each well and incuba ed a 37 ºC o 45 min. A e washing he
144
pla e 3 imes wi h PBS-T and deionised wa e , 100 µL pe well o an i-digoxigenin
145
an ibody labelled wi h ho se adish pe oxidase (an i-Dig-HRP) solu ion in PBS-T
146
(1:2000) we e dispensed and incuba ed a oom empe a u e o 25 min. A e a washing
147
s ep wi h PBS-T and deionised wa e , 100 µL o TMB solu ion (0.25 g L-1 o 3, 3’, 5,
148
5’- e ame hylbenzidine and 0.002 M o hyd ogen pe oxide in ci a e bu e , pH 5.5)
149
we e dispensed and incuba ed a oom empe a u e o 10 min. Finally, he eac ion was
150
s opped wi h 50 µL o 2.5 M sulphu ic acid and abso bance was measu ed a 450 nm
151
( e e ence wa eleng h: 650 nm) wi h a mic o i e pla e eade (Wallac, model Vic o
152
1420 mul ilabel coun e , Finland). A sample was conside ed posi i e when he op ical
153
esponse was highe han he cu -o alue.
154
155
2.4 PCR-ELISA
156
PCR mix u es (25 μL) con ained 15 ng o ex ac ed genomic DNA, 1× T is-KCl
157
bu e (100 mM T is-HCl, 500 mM KCl, pH 8.3), 2 mM MgCl2, 200 µM dNTPs, 1.25
158
uni s o Taq DNA polyme ase (Roche, Ge many) and 400 nM o each 5’-digoxigenin
159
labelled p ime (Table 1). Reac ions we e ca ied ou in a TC-400 he mocycle (Bibby
160
Scien i ic, S a o dshi e, UK) and applying he he mal p og amme: dena u a ion (95
161
6
ºC, 5 min) ollowed by 40 cycles o dena u a ion (95 ºC, 30 s), annealing (62 ºC, 30 s)
162
and elonga ion (72 ºC, 30 s), wi h a inal elonga ion s ep (72 ºC, 5 min). The
163
immunoassay was pe o med as is desc ibed abo e.
164
165
2.5 Compa ison wi h o he de ec ion echniques
166
Ampli ica ion p oduc s we e checked by elec opho esis on a 3% (w/ ) aga ose
167
gel a 110 V and oom empe a u e. Gels we e s ained o 30 min wi h 0.5× TBE bu e
168
(T is/Bo a e/EDTA) con aining luo opho e Real-Sa e (Real Labo a o ies, Spain) a
169
0.01% ( / ), and bands we e isualised wi h a UV ansillumina o . P oduc size was
170
de e mined by compa ison wi h a 50 bp ladde (Fe men as, Li huania). Ampli ica ion
171
yields we e calcula ed om he luo escence measu emen s wi h SYBR-Sa e a 0.01%
172
( / ) in a mic o i e pla e eade .
173
174
2.6 Compa ison o de eloping s a egies
175
O he de eloping immunoassay s eps we e also es ed independen ly. o-
176
Phenylenediamine dihyd ochlo ide (OPD) was used as an al e na i e colo ime ic
177
subs a e o TMB. The addi ion o digoxigenin-dUTPs (35 µM) in he RPA mix u es
178
was an al e na i e o ob ain labelled ampli ied p oduc s. Th ee an igen-an ibody
179
ecogni ion assays we e also compa ed. The i s app oach was based on digoxigenin-
180
labelled p ime s, an an i-digoxigenin an ibody p oduced in sheep (an i-Dig) as a
181
p ima y an ibody a he 1/20,000 dilu ion, and an i-sheep conjuga ed wi h ho se adish
182
pe oxidase (an i-sheep-HRP) as a seconda y an ibody a he 1/4,000 dilu ion. The
183
second app oach used Cy5-labelled p ime s, an an i-Cy5 an ibody p oduced in mouse
184
(an i-Cy5) a he 1/2,000 dilu ion, and an an i-mouse conjuga ed wi h ho se adish
185
pe oxidase (an i-mouse-HRP) a he 1/500 dilu ion. The hi d app oach was based on
186
Dig-labelled p ime s, sheep an i-Dig an ibody as a p ima y an ibody a he 1/5,000
187
dilu ion, and an i-sheep conjuga ed wi h alkaline phospha ase (an i-sheep-AP) as a
188
seconda y an ibody dilu ed a 1/250, using he ni o-blue e azolium/5-b omo-4-chlo o-
189
3'-indolyphospha e solu ion (BCIP/NBT) as he colo ime ic subs a e. Da a analysis
190
was pe o med wi h he s a is ical package SPSS o Windows, . 16.0
191
192
7
3. Resul s and discussion
193
3.1 Adap a ion o he RPA p o ocol
194
The eac ion condi ions (p ime concen a ions, empe a u e and ime) we e s udied o
195
achie e he same ampli ica ion condi ions o en analy es: hazelnu , peanu , soybean,
196
and maize seeds, oma o ui , P35S and TNOS om he CRM, and pu e cul u es o
197
Salmonella spp., C onobac e spp., and Fusa ium spp. The op imal RPA condi ions
198
we e 480 nM o he o wa d and e e se p ime s, and incuba ion a 40 ºC o 40 min. A
199
single p o ocol was achie ed o he pa allel ampli ica ion o all es ed analy es, which
200
conside ably cu he o al analysis ime. Ampli ied p oduc s we e cha ac e ised by
201
aga ose gel elec opho esis o p oduc size de e mina ion. RPA eac ions gene a ed he
202
p edic ed p oduc leng h, acco ding o he p oposed p ime s, hese being: 109 bp o
203
hazelnu , 82 bp o peanu , 81 bp o soybean, 92 bp o oma o, 136 bp o maize, 123
204
bp o P35S, 118 o TNOS, 152 bp o Salmonella spp., 190 bp o C onobac e spp.,
205
and 180 bp o Fusa ium spp.
206
207
3.2 Hyb idisa ion assays. In eg a ion o RPA and ELISA
208
Se e al ac o s in luence hyb idisa ion e iciency o he speci ic p obe
209
immobilised on he mic opla e, and la e he de ec ion esponse. Fo his pu pose, mul i-
210
a iable expe imen al designs we e made o op imise he main pa ame e s du ing he
211
assays.
212
The hyb idisa ion p ocess on solid suppo s, including polys y ene used in
213
mic opla e wells, depends on p obe co e age densi y. An indi ec immobilisa ion
214
eac ion (s ep a idin/bio in-labelled p obe) was chosen. To ha end, coa ing condi ions
215
we e op imised by a ying he s ep a idin concen a ion om 0.002 o 2 mg L-1, and
216
he p obe concen a ion om 0.2 o 200 nM, and 0.2 mg L-1 o s ep a idin and he 20
217
nM p obe we e selec ed (Figu e 1A). A s ep a idin concen a ion abo e 0.2 mg L-1
218
inc eased he signal, bu d as ically educed assay ep oducibili y. P obe co e age was
219
calcula ed using double labelled oligonucleo ides (5’-bio in and 3’-Cy5) and a
220
homemade su ace luo escence eade . P obe densi y was 0.08 mol mm-2.
221
Addi ionally, he hyb idisa ion yield changed acco ding o empe a u e. Hyb idisa ion
222
ime was es ed e e y 10 min o 1 h, and he empe a u e ange was 25-50 ºC. The
223
maximum signal was achie ed a e 45 min a 37 ºC (Figu e 1B and 1C).
224
8
The e ec o he eac ion olume (12.5-200 µL/well) and p oduc dilu ion (1/50 o
225
1/5,000) was also e alua ed (Figu e 1D). The bes esul s we e ob ained a he 1/100
226
dilu ion o ampli ica ion p oduc and using 100 µL as he eac ion olume. Highe
227
concen a ions o ampli ica ion p oduc educed he signal. This e ec can be explained
228
by he p esence o Ca bowax 20M (5%) in he RPA bu e . No o he e ec s o
229
ampli ica ion eagen s o he washing p o ocol we e obse ed, so i was unnecessa y o
230
pe o m u he ea men s wi h he RPA p oduc s in he p oposed me hod. A e es ing
231
some solu ions (wa e , SSC, PBS-T, and PBS) and cycles (1-5), h ee cycles wi h PBS-
232
T and one cycle wi h deionised wa e we e chosen as he app op ia e washing p o ocol.
233
234
3.3 Op imisa ion o he de ec ion s ep
235
The ELISA assays based on he ecogni ion digoxigenin-labelled RPA p oduc s
236
by an an i-Dig-HRP an ibody we e op imised by analysing he DNA ex ac ed om
237
hazelnu , soybean seeds and ansgenic maize. The highes signal was achie ed a he
238
1/2,000 dilu ion, and no signi ican signal imp o emen was accomplished a highe
239
concen a ions (Figu e 1E). The an ibody incuba ion ime was op imised, wi h he bes
240
esul s ob ained a 25 min (Figu e 1F). Th ee PBS-T washing s eps and u he
241
deionised wa e ising we e su iced o elimina e any excess eagen s.
242
Fo he immunoenzyma ic de ec ion o he hyb idisa ion complex, a colo ime ic
243
eac ion ( = 450 nm, backg ound = 650 nm) was employed by compa ing wo common
244
HRP subs a es, 3,3’,5,5’- e ame hylbenzidine (TMB) and o-phenylenediamine
245
dihyd ochlo ide (OPD), a di e en concen a ions up o 2 g L-1. As no ema kable
246
di e ences we e obse ed du ing he achie ed analy ical pe o mances be ween bo h
247
subs a es, he use o TMB a 0.25 g L-1 was selec ed because i o e s ad an ages, such
248
as low cos , easy manipula ion, g ea e s abili y and lowe oxici y [8].
249
Al e na i e s a egies o he digoxigenin-labelled p ime s/an i-Dig-HRP/TMB
250
sys em we e adop ed and compa ed o con en ional me hods: elec opho esis and in-
251
well- luo escence by he SYBR-Sa e DNA s ain (Figu e 2). Fi s ly, digoxigenin-dUTPs
252
in he PCR mix u es we e es ed as an example o nucleo ide labelling. This op ion,
253
ei he combined o no wi h digoxigenin-labelled p ime s, inc eases assay sensi i i y,
254
bu a a sligh ly highe cos -by- eac ion [8]. Secondly, he immunoassay can be
255
pe o med using di e en combina ions o p ima y and seconda y an ibodies. Two
256
app oaches we e es ed: digoxigenin-labelled p ime s/sheep an i-Dig/an i-sheep-HRP
257
and Cy5-labelled p ime s/mouse an i-Cy5/an i-mouse-HRP. By keeping digoxigenin as
258
15
[20] Commission Regula ion (EU) No 619/2011 o 24 June 2011 laying down he
402
me hods o sampling and analysis o he o icial con ol o eed as ega ds p esence o
403
gene ically modi ied ma e ial o which an au ho isa ion p ocedu e is pending o he
404
au ho isa ion o which has expi ed. O J Eu Union L 268:24–28.
405
[21] Commission Regula ion (EC) No 2073/2005 o 15 No embe 2005 on
406
mic obiological c i e ia o oods u s. O J Eu Union L 338:1–26.
407
[22] T. A nandis-Cho e , S. Mo ais, L.A. To ajada-Gena o, R. Puchades, A.
408
Maquiei a, J. Be ganza, G. Olaba ia, Talan a, 101 (2012) 405-412.
409
[23] C. G imm, R. Geisen, Le . Appl. Mic obiol. 26 (1998) 456-462.
410
[24] Commission Regula ion (EC) No 1881/2006 o 19 Decembe 2006 se ing
411
maximum le els o ce ain con aminan s in oods u s. O J Eu Union L 364:5–24.
412
[25] L. Yang, A. Pan, J. Jia, J. Ding, J. Chen, H. Chen, C. Zhang, D. Zhang, J. Ag ic.
413
Food Chem. 53 (2005) 183-190.
414
[26] In e na ional O ganiza ion o S anda diza ion. 21570:2005 Foods u s–Me hods
415
o analysis o he de ec ion o gene ically modi ied o ganisms and de i ed p oduc s–
416
quali a i e nucleic acid based me hods. Gene a, Swi ze land: In e na ional O ganiza ion
417
o S anda diza ion, 2005.
418
[27] In e na ional O ganiza ion o S anda diza ion. 21569:2005 Foods u s–Me hods
419
o analysis o he de ec ion o gene ically modi ied o ganisms and de i ed p oduc s–
420
quali a i e nucleic acid based me hods. Gene a, Swi ze land: In e na ional O ganiza ion
421
o S anda diza ion, 2005.
422
[28] Wo ld Heal h O ganiza ion. 2004. In e na ional Cou se: Epidemiology o
423
oodbo ne diseases. Au onomous Uni e si y o Yuca an, Me ida, Mexico. pp. 25-36.
424
[29] R. Köppel, F. an Velsen-Zimme li, T. Buche , Eu . Food Res. Technol. 235
425
(2012) 843-853.
426
[30] P.P. Zhou, J.Z. Zhang, Y.H. You, Y.N. Wu, Biomed. En i on. Sci. 21 (2008) 53-
427
62.
428
[31] Regula ion (EC) No 1830/2003 o he Eu opean Pa liamen and o he Council o
429
22 Sep embe 2003 conce ning he aceabili y o ood and eed p oduc s p oduced om
430
gene ically modi ied o ganisms and amending Di ec i e 2001/18/EC. O J Eu Union L
431
166:9–15.
432
433
434

16
FIGURE CAPTIONS
435
Figu e 1. RPA-ELISA op imiza ion: e ec o di e en expe imen al a iables on op ical in ensi y
436
(A) Coa ing condi ions (s ep a idin and p obe concen a ions); (B) Hyb idisa ion ime; (C) Hyb idisa ion
437
empe a u e; (D) Hyb idisa ion solu ion (dilu ion RPA solu ion and o al olume); (E) De eloping agen
438
dilu ion (dilu ion o an i-Dig-HRP an ibody); (F) De eloping ime (an ibody incuba ion). Signal
439
co esponds o 1.5 ng o a ge genomic DNA.
440
441
Figu e 2. Compa ison o de ec ion s a egies o RPA p oduc s. B -p obe: bio inila ed p obe; an i-
442
Dig: an i-digoxigenin an ibody p oduced in sheep; an i-sheep: an i-sheep an ibody; an i-Cy5: an i-Cy5
443
an ibody p oduced in mouse; an i-mouse: an i-mouse an ibody; HRP ho se adish pe oxidase; AP: alkaline
444
phospha ase. Signal co esponds o he sensi i i y o each o ma (calcula ed in CFU/mL), de e mined by
445
analysing se ially dilu ed DNA ex ac s om Salmonella spp.
446
447
Figu e 3. Naked-eye esul s o comme cial ood samples in mic opla es. Each sample ( ow) is
448
es ed o each analy e (columns): (1) hazelnu ; (2) peanu ; (3) soybean; (4) oma o; (5) maize; (6) P35S;
449
(7) TNOS; (8) Samonella spp.; (9) C onobac e spp.; (10) Fusa ium spp. Highligh ed ec angles indica e
450
posi i e samples (abso bance > cu -o alue).
451
452
TABLE CAPTIONS
453
Table 1. The p ime s, p obes, and con ol sequences used o ampli ica ion p ocedu es
454
455
Table 2. Compa ison o limi s o de ec ion and ep oducibili y ob ained by RPA-ELISA and PCR-
456
ELISA
457
458
Table 3. Sc eening esul s o he analy es in comme cial ood samples analysed by RPA-ELISA
459
460
17
GRAPHICAL ABSTRACT
461
462
S ep a idin
Conjuga ed an ibody
P obe
Ta ge DNA
ISOTHERMAL
AMPLIFICATION
RPA-ELISA
18
Figu e 1.
463
464
0 0.002 0.02 0.2 2
0
0.2
2
20
200
S ep a idin (mg L-1)
P obe (nM)
A
0.8-1.0
0.6-0.8
0.4-0.6
0.2-0.4
0.0-0.2
Abs 450/650 nm
B
0.0
0.2
0.4
0.6
0.8
1.0
010 20 30 40 50 60
Time (min)
Abs 450/650 nm
C
0.0
0.2
0.4
0.6
0.8
1.0
25 30 35 40 45 50
Tempe a u e (ºC)
Abs 450/650 nm
0 12.5 25 50 100 150 200
1/5000
1/2500
1/1000
1/500
1/100
1/50
Volume (µL)
RPA p oduc dilu ion
D
0.8-1.0
0.6-0.8
0.4-0.6
0.2-0.4
0.0-0.2
Abs 450/650 nm
E
0.0
0.2
0.4
0.6
0.8
1.0
1/20000
1/10000
1/8000
1/6000
1/4000
1/2000
1/1000
an i-Dig-HRP dilu ion
Abs 450/650 nm
F
0.0
0.2
0.4
0.6
0.8
1.0
0 5 10 15 20 25 30
De eloping ime (min)
Abs 450/650 nm
465
466
19
Figu e 2
467
468
20
Figu e 3
469
1 2 3 4 5 6 7 8 9 10
Muesli cookies
Ke chup
Soup
Powde ed in an o mula
Nega i e con ol
Posi i e con ol
1 2 3 4 5 6 7 8 9 10
Muesli cookies
Ke chup
Soup
Powde ed in an o mula
Nega i e con ol
Posi i e con ol
470
471

21
Table 1.
472
Ta ge
Sequence 5’-3’
Tm
(ºC)
Amplicon
size (bp)
Re e ence
Hazelnu
FP
Dig-ACTACATAAAGCAAAAGGTTGAAG
53.5
109
[4]
Co a1 gene
RP
TCGTAATTGATTTTCTCCAGTTTG
55.2
P obe
B nTg-TTTTTCGGACAAAGCATCGCCTTCAATCA
67.1
Peanu
FP
Dig-CTAGTAGCCCTCGCCCTTTT
59.9
82
[4]
A a h2 gene
RP
GGCATCTTCTGTCTCCTTGG
59.8
P obe
B nTg-TTTTTAGTTCCCACTGCTGCCTC
62.6
Soybean
FP
Dig-TCCACCCCCATCCACATTT
59.2
81
[4]
Le gene
RP
GGCATAGAAGGTGAAGTTGAAGGA
58.8
P obe
B nTg-TTTTTTTTTTCGAAGCTGGCAACGCTACCGGTT
74.1
Toma o
FP
Dig-AGACCACGAGAACGATATTTGC
66.8
92
[25]
La 52 gene
RP
TTCTTGCCTTTTCATATCCAGACA
58.4
P obe
B nTg-TTTTTACTCTCTTTGCAGTCCTCCCTTGGG
57.6
Maize
FP
Dig-CGTCGTTTCCCATCTCTTCCTCC
64.2
136
[26]
adh 1 gene
RP
CCACTCCGAGACCCTCAGTC
63.5
P obe
B nTg-TTTTTCCTCACCAGTTACGAAACCAATCGATCCAA
67.1
GMO p omo e
FP
Dig-CCACGTCTTCAAAGCAAGTGG
59.8
132
[27]
35S gene
RP
TCCTCTCCAAATGAAATGAACTTCC
59.7
P obe
B nTg-TTTTTTTATATAGAGGAAGGGTCTTGCGAAGGATA
64.8
GMO e mina o
FP
Dig-GCATGACGTTATTTATGAGATGGG
59.3
118
[27]
NOS gene
RP
GACACCGCGCGCGATAATTTATCC
64.4
P obe
B nTg-TTTTTTTTGCGCGCTATATTTTGTTTTCTATCGCG
64.8
Salmonella spp.
FP
Dig-TACCAAAGCTAAACGCGCAGCT
62.1
152
[8]
hns gene
RP
TGATCAGGAAATCTTCCAGTTGC
61.1
P obe
B nTg-TTTTTTTTTTTTTGATTACAGCCGGTGTACGACCCT
75.9
C onobac e spp.
FP
Dig-GTTGGATCACCTCCTTACCTGC
64.2
190
[8]
16S-23S DNA gene
RP
AGTTAAACCTCTTCAACTCCTG
58.4
P obe
TGTGAGCACGCGAGGTTGTATCTTGCA-TTTTTTTTTT-B nTg
64.0
Fusa ium spp.
FP
Dig-CCGAGTTTACAACTCCCAAA
62.7
180
[5]
ITS 1 gene
RP
ACAGAGTTTAGGGGTCCTCT
58.4
P obe
B nTg-TTTTTTTTTTTTACCGGGAGCGGGCTGAT
67.4
Posi i e con ol
P obe
Dig-TTTTTTTTTTTTTTTGTCATGGGCCTCGTGTCGGAAAACC-B nTg
81.0
Nega i e con ol
P obe
B nTg-ACCGTCGCGCACTATCTGATTTCAAA
73.3
FP: o wa d p ime , RP: e e se p ime , Dig: digoxigenin-labelled, B n-Tg: bio in labelled
473
474
22
Table 2.
475
RPA-ELISA
PCR-ELISA
Hazelnu (µg g-1)*
1.29
5.80
Peanu (µg g-1)
11.21
13.27
Soybean (µg g-1)
2.01
1.47
Toma o (µg g-1)
9.45
6.63
Maize (µg g-1)
14.36
2.00
Limi o de ec ion
P35S (µg g-1)
8.36
1.24
TNOS (µg g-1)
19.74
6.69
Salmonella spp. (CFU mL-1)
6.00
5.00
C onobac e spp. (CFU mL-1)
13.00
12.00
Fusa ium spp. (µg g-1)
5.93
31.96
Mean
Rep oducibili y** (%)
In a-day
1.4 - 6.6
3.4 - 7.2
In e -day
7.9 - 11.3
8.5 - 14.5
* µg g-1 e e s o µg o analy e pe g o ood
476
** Rep oducibili y was calcula ed om he samples con aining 0.1 % o analy e (n=3)
477
478
23
Table 3.
479
Decla ed analy ea / De ec ed analy ec
Spiked analy eb / De ec ed analy ec
Food
Hazelnu
Peanu
Soybean
Toma o
Maize
P35S
TNOS
S. spp
C. spp
F. spp
Muesli cookies
+ / ++
- / nd
+ / +
- / nd
± / +
<0.9% / +
<0.9% / +
- / nd
- / nd
10% / +++
Chocola e wa e
± / +
+ / +
± / +
- / nd
- / nd
<0.9% / nd
<0.9% / nd
4·101 / +
- / nd
- / nd
Ke chup
- / nd
- / nd
- / nd
+ / ++
+ / ++
<0.9% / nd
<0.9% / nd
4·103 / ++
- / nd
0.1% / +
Feed
- / nd
- / nd
- / nd
- / nd
+ / +
+ / ++
+ / +
4·104 / +++
- / nd
1% / ++
Toma o
- / nd
- / nd
- / nd
+ / +++
- / nd
+ / ++
+ / ++
4·103 / ++
4·102 / ++
- / nd
Baby ood
- / nd
- / nd
- / nd
- / nd
+ / +++
<0.9% / nd
<0.9% / nd
4·104 / +++
- / nd
10% / +++
Soup
- / nd
- / nd
± / +
- / nd
- / nd
<0.9% / nd
<0.9% / nd
4·102 / ++
4·102 / ++
- / nd
Skimmed powde ed milk
- / nd
- / nd
- / nd
- / nd
- / nd
<0.9% / nd
<0.9% / nd
4·102 / ++
4·104 / +++
- / nd
Powde ed in an o mula
- / nd
- / nd
- / nd
- / nd
- / nd
<0.9% / nd
<0.9% / nd
4·104 / +++
4·103 / +++
- / nd
CRM (RRS 5%)
- / nd
- / nd
+ / +++
- / nd
- / nd
+ / ++
+ / +
- / nd
- / nd
- / nd
CRM (B 11 Maize 5%)
- / nd
- / nd
- / nd
- / nd
+ / +++
+ / ++
+ / +
- / nd
- / nd
- / nd
Swee co n
- / nd
- / nd
- / nd
- / nd
+ / +++
<0.9% / nd
<0.9% / nd
- / nd
- / nd
1% / ++
a Decla ed: + analy e lis ed; - analy e no lis ed; ± may con ain ace le els; <0.9% labelling no equi ed (GMO-EU egula ion).
480
b Spiked analy e co espond o Salmonella spp. (S. spp), C onobac e spp. (C. spp), and Fusa ium spp. (F. spp).
481
c Used code: +, de ec ed a low le el; ++, de ec ed a medium le el; +++, de ec ed a high le el; nd, non de ec ed.
482
483