167
Phy opa hologia Medi e anea (2017), 56, 1, 167−174
www. up ess.com/pm ISSN (p in ): 0031-9465
Fi enze Uni e si y P ess ISSN (online): 1593-2095
DOI: 10.14601/Phy opa hol_Medi e -20293
Co esponding au ho : T. Yli-Ma ila
E-mail: [email p o ec ed]
© 2017 Au ho (s). This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC-BY-4.0),
which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
TOOLS FOR FUSARIUM MYCOTOXIN REDUCTION IN FOOD AND FEED CHAINS
RESEARCH PAPERS
Di e en g ain g inding me hods a ec de ec ion o Fusa ium
g aminea um DNA and myco oxins
Tapani YLI-MATTILA1, Sa i RÄMÖ2, Taha HUSSIEN1,4, Ma ion RAUVOLA5, Veli HIETANIEMI3 and Jukka KAITARANTA5
1 Molecula Plan Biology, Depa men o Biochemis y, Uni e si y o Tu ku, FI-20014 Tu ku, Finland
2 Na u al Resou ces Ins i u e Finland (Luke), Managemen and P oduc ion o Renewable Resou ces, FI-31600 Jokioinen, Finland
3 Na u al Resou ces Ins i u e Finland (Luke), Bio-Based Business and Indus y, FI-31600 Jokioinen, Finland
4 Myco oxins Lab, Depa men o Toxins and Con aminan s, Na ional Resea ch Cen e , Cai o, Egyp
5 Tu ku Uni e si y o Applied Sciences, FI-20520 Tu ku, Finland
Summa y. Fusa ium g aminea um is he mos impo an deoxyni alenol (DON) p oducing species in no he n Eu-
ope and Asia. The highes DON le els in Finland ha e been ound in oa g ain, and DNA le els o F. g aminea um
a e in ag eemen wi h DON le els, when DON is measu ed by acc edi ed GC-MS. In addi ion o DON, 3ADON is
p esen in Finnish g ain samples. La ge a ia ions in DON con en and amoun s o F. g aminea um DNA, and poo
coe icien o de e mina ion (R2) be ween hese, ha e been de ec ed in oa g ain when he RIDA®QUICK SCAN ki
esul s o DON con en ha e been used. This s udy con i med ha he coe icien o de e mina ion was usually
less when DNA o DON we e ex ac ed om oa lou , which was no g ound wi h 0.8 mm o 1 mm sie es. DON
le els ob ained wi h he Rida®Quick me hod we e usually highe han hose ob ained wi h acc edi ed GC-MS
in Finnish oa , ba ley and whea samples. The homogeniza ion o he oa lou by sie ing is he e o e likely o be
connec ed o he a ia ions in DON de ec ion. Amoun s o F. g aminea um DNA and DON close o legisla i e limi s
should be econ i med wi h acc edi ed quan i a i e analyses.
Key wo ds: oa , ba ley, whea , myco oxins, qPCR.
In oduc ion
Fusa ium head bligh (FHB) was i s desc ibed
in Eu ope in England in 1884 (Pa y e al., 1995). By
1924, FHB was al eady ound in England, Russia,
Sweden, F ance, I aly, Ge many, Holland and No -
way. Acco ding o Sundheim e al. (2013), he i s F.
g aminea um isola e in No way was ound om oa
in 1911, while in Finland he ungus was epo ed in
1932 (Rainio, 1932). A p esen , FHB is common in
all Eu opean coun ies p oducing whea , maize, and
o he ce eals such as ba ley and oa .
Fusa ium g aminea um sensu s ic o, oge he wi h
o he closely ela ed species o he F. g aminea um spe-
cies complex, is he mos common cause o FHB in
whea , ba ley, oa and o he small g ain ce eals in mos
pa s o he wo ld. The main species o F. g aminea-
um species complex in Eu ope is F. g aminea um sensu
s ic o, which is also he mos impo an deoxyni ale-
nol (DON) p oduce in mos coun ies in Eu ope (Pas-
quali e al., 2016; Yli-Ma ila and Gagkae a, 2016).
Fusa ium g aminea um DNA le els in oa a e as-
socia ed wi h DON le els, when DON is measu ed
by gas ch oma og aphy-mass spec ome y (GC-
MS). When he Rida® Quick La e al Flow es esul s
o DON con en we e compa ed o DNA le els o F.
g aminea um, he ela ionships be ween he wo we e
much less dis inc . The homogeniza ion o he oa
lou du ing g inding o g ain and passage h ough
a 1 mm sie e is likely o be connec ed o his a ia-
ion (Yli-Ma ila e al., 2013).
In he No dic coun ies, high le els o F. g aminea-
um and DON ha e been ound especially in oa g ain,
Phy opa hologia Medi e anea
168
T. Yli-Ma ila e al.
while in no h-wes e n Russia F. g aminea um was
ound only a e 2003 (Yli-Ma ila e al., 2009; Yli-Ma i-
la and Gagkae a, 2010; F edlund e al., 2013; Sundheim
e al., 2013). High g ain in es a ion le els o F. g aminea-
um and amoun s o DON ha e caused se ious losses
in ge mina ion a many ins ances e.g. in No way (Ki-
men Seed Labo a o y, 2009). Inc eased F. g aminea um
and DON le els may be associa ed wi h clima e change
( an de Fels-Kle x e al., 2012). These e ec s ha e led
he equi emen o seed impo s in o No way.
Recen ly, F. g aminea um has been sp eading
no hwa d in Eu ope (e.g., Waalwijk e al., 2003; Ni-
cholson e al. 2003; S epien e al., 2008; Nielsen e al.,
2011; Talas e al., 2011; Somma e al., 2014; Sup oniene
e al., 2010, 2016) displacing he closely ela ed F. cul-
mo um. This shi may be due o changing ag icul-
u al p ac ices, clima e change and inc eased maize
c opping. In Finland, F. g aminea um was epo ed in
ce eals in 1932 (Rainio, 1932) and in he 1960s (Uo i
and Ylimäki, 1974), and his ungus has been p esen
since hen (Ylimäki e al., 1979; Eskola e al., 2001).
In no h-wes e n Russia F. g aminea um was ound
mo e ecen ly (Yli-Ma ila e al., 2009). Fusa ium
g aminea um is also p esen in No way, Sweden and
Es onia (Yli-Ma ila e al., 2011). Acco ding o Sund-
heim e al. (2013) and Aamo e al. (2015), F. g amine-
a um has become he dominan DON-p oducing
species in No way since 2000. Also in Finland, F.
g aminea um is now he main DON-p oduce in ce e-
als (Yli-Ma ila, 2010, Hie aniemi e al., 2016)
The 3ADON chemo ype o F. g aminea um is
dominan in mos no he n a eas, while he 15ADON
chemo ype is p edomina ing in Cen al and sou h-
e n Eu ope (Pasquali e al., 2016). The no h-wes e n
Russian isola es belong o he 3ADON chemo ype,
like Finnish isola es and unlike mos isola es om
sou he n Russia. This sugges s ha he no h-wes -
e n Russian F. g aminea um popula ions may ha e
o igina ed om Finland, while he sou he n Russian
popula ions may be mo e closely ela ed o he cen-
al and sou he n Eu opean 15ADON popula ions,
as sugges ed by Yli-Ma ila (2010).
Se e al immunological me hods ha e been de el-
oped o es ima ion o DON le els in ou ine analyses
ca ied ou by ood labo a o ies. Howe e , an ibodies
may show c oss- eac i i y wi h analogous compounds.
Thus, DON can be o e es ima ed, wi h 15ADON,
3ADON, DON-3β-glucoside and o he DON de i a-
i es, which a e masking compounds o med by plan
de oxi ica ion ac i i y (Lemmens e al., 2005; Audenae
e al., 2013) , while ma ix e ec s can lead o o e - o
unde -es ima ion o myco oxins (Aamo e al., 2012).
The aim o he p esen s udy was o in es iga e
DON le els in Finnish ce eal g ain, ob ained wi h ac-
c edi ed GC-MS and Rida®Quick me hods, o com-
pa e hese wi h F. g aminea um DNA le els, and also
measu e e ec s o g ain g inding me hods on DON
and F. g aminea um DNA le els.
Ma e ials and me hods
Fusa ium isola e
The single spo e isola e G243 o F. g aminea um
was used as he s anda d in qPCR assays, as de-
sc ibed by Yli-Ma ila e al. (2008, 2011).
Myco oxin analyses
Oa g ain samples (21 samples) o he yea 2013
we e a pa o he o al numbe o samples ana-
lyzed o DON, 3ADON, 15ADON and o he i-
cho hecenes a MTT Ag i ood Resea ch Finland
(Na u al Resou ces Ins i u e Finland, FI-31600 Jo-
kioinen, Finland). These we e selec ed om 125 oa
samples, so ha in 19 samples had DON le els g ea -
e han 1,750 ppb, one had 1,100 ppb DON and one
had less han 200 ppb. In addi ion, he DON le els in
20 oa samples om he yea 2013 we e es ima ed us-
ing Rida® Quick Scan myco oxin es s (R-Biopha m
AG). These samples we e selec ed so ha 15 con-
ained mo e han 1,750 pbb o DON and i e samples
had less han 1,750 ppb. A MTT, he icho hecenes
we e ex ac ed and analyzed wi h an acc edi ed GC-
MS me hod, as desc ibed by Hie aniemi e al. (2004)
and Yli-Ma ila e al. (2011). The g ain lou was p o-
duced using a Falling numbe hamme mill KT-120
(Kone eollisuus Oy) using a 1 mm sie e in o de o
ob ain homogenous samples and o achie e good
epea abili y in myco oxin and DNA measu emen s.
The g ain samples om a majo milling company
we e used o Rida®Quick measu emen o DON
and hey we e g ound using a sepa a e mill wi hou
a sie ing p ocedu e p io o oxin measu emen .
DNA ex ac ion
A he Uni e si y o Tu ku, he 21 oa g ain sam-
ples om MTT and he 20 samples om he milling
company used o myco oxin analyses we e g ound
169
Vol. 56, No. 1, Ap il, 2017
G ain g inding and Fusa ium g aminea um de ec ion
o DNA ex ac ion using a mill (K ups KM75 Co ee
G inde ) wi hou sie ing as desc ibed by Yli-Ma ila
e al. (2013). DNA was ex ac ed om g ound g ain
samples and om he F. g aminea um isola e G243
s anda d using he GenElu e™ Plan Genomic DNA
ki (Sigma-Ald ich), as desc ibed by Yli-Ma ila e al.,
(2008, 2009, 2013).
Quan i a i e PCR
The TMFg12 p ime s and p obe ha e been de-
signed o he F. g aminea um speci ic RAPD-PCR
p oduc (Yli-Ma ila e al., 2008). An IQTM5 Real-
Time PCR De ec ion Sys em (Bio-Rad) was used o
unning qPCR samples. Amoun s o Fusa ium spe-
cies DNA in g ain samples we e de e mined as o al
DNA quan i ied by a Qubi luo ome e (In i ogen),
as desc ibed by Yli-Ma ila e al. (2011).
Rida®Quick DON analyses
Oa (30), whea (20) and ba ley (20) samples om
di e en loca ions in Finland we e analyzed using
he Rida®Quick DON me hod a Tu ku Uni e si y o
Applied Sciences (TUAS), and he esul s we e com-
pa ed wi h he acc edi ed GC-MS me hod desc ibed
abo e. The ep oducibili y o he me hod was es ed
wi h six pa allel samples om he h ee ce eals, wi h
h ee di e en DON le els. G ound oa samples (12)
we e also ob ained om an indus ial labo a o y
(Labo a o y 1), and 12 g ound whea samples we e
ob ained om a second indus ial labo a o y (Labo-
a o y 2).
G ain samples o compa ing he wo me h-
ods o measu ing DON le els we e g ound using
h ee mills. In Labo a o y 1 hey we e g ound using
labo a o y mill KT 30 (Kone eollisuus OY) wi hou
sie ing, while in he Labo a o y 2 he samples we e
g ound by labo a o y mill 3100 Falling numbe wi h
a sie e o 0.8 mm. A MTT, a labo a o y Falling num-
be hamme mill wi h a 1 mm sie e was used as de-
sc ibed abo e.
Each g ound g ain sample (1 g) was placed in a
Falcon ube and 15 ml o Rida®Quick DON ex ac-
ion bu e was added. The mix u e was manually
shaken o 3 min be o e cen i uga ion o 10 min
(3,500 g). The clea supe na an (100 μL) was pipe -
ed on o he applica ion a ea o he es s ip and he
esul was ead a e 5 min.
S a is ical analyses
R2 (coe icien o de e mina ion), eg ession slope
and P (signi icance o eg ession slope) we e calcu-
la ed using he SigmaPlo e sion 12.0 (SPSS Inc.).
The o iginal DNA and oxin concen a ions we e
ans o med o loga i hmic alues [1 + lg (x)] o ob-
ain no mal dis ibu ion o hese pa ame e s. Sam-
ples gi ing concen a ions less han 0.5 mg kg-1 o
g ea e han 5.5 mg kg-1 wi h Rida®Quick we e ex-
cluded om he analyses, because hese alues we e
ou side he linea pa o he de ec ion.
The ep oducibili y be ween amoun s o DON
es ima ed wi h Rida®Quick in oa g ain samples
was es ed using h ee samples wi h DON le els o
1.4, 1.7 and 2.1 mg kg-1 (n = 6, Table 1). Comme cial
s anda d (Check sample Deoxyni alenol in whea .
Rome Labs. 1.431 ± 0.2566 mg kg-1) was also used.
In ba ley, he DON le els we e 0.81, 1.30 and 1.60
mg kg-1, while in whea hey we e 0.79, 1.00 and 1.40
mg kg-1. Rida®Quick esul s we e compa ed o ac-
c edi ed GC-MS esul s using eg ession analysis,
and pai ed - es s we e applied o e eal he 95 %
signi icance le el. Rela i e s anda d de ia ion (RSD
%) was also calcula ed.
Table 1. The ep oducibili y o DON de ec ion esul s
(mean + SE o six DON ex ac ions and DON measu e-
men s o each g ound g ain sample ob ained om MTT)
in oa , ba ley and whea g ain samples wi h h ee di e en
DON con en s.
G ain MTT esul
(mg kg-1)
Rida®Quick esul
(mg kg-1 ± SE)
RSD
%
Oa 1.40 1.69 ± 0.04 5.27
1.70 1.98 ± 0.07 8.51
2.10 2.32 ± 0.09 9.87
Ba ley 0.81 1.80 ± 0.16 22.3
1.30 2.30 ± 0.12 12.7
1.60 2.32 ± 0.150 15.8
Whea 0.79 1.32 ± 0.06 22.6
1.00 1.59 ± 0.15 12.0
1.40 2.11 ± 0.10 10.3
Phy opa hologia Medi e anea
170
T. Yli-Ma ila e al.
Resul s
In he ep oducibili y analysis be ween he
Rida®Quick and GC-MS de ec ion me hods, mos
esul s ga e RSD pe cen ages equal o o less han
20, especially when he DON le els we e close o o
abo e he allowed limi (Table 1).
The co ela ion be ween F. g aminea um DNA and
DON le els in Finnish oa samples om he yea 2013
was g ea e (R2 = 0.76), when he DNA was ex ac ed
and measu ed om g ain samples g ound a MTT
using a mill wi h a sie e. When he F. g aminea um
DNA was measu ed om he same g ain samples
g ound a he Uni e si y o Tu ku, using a me hod
lacking he sie e, he co ela ion was less (R2 = 0.31,
Figu e 1). The R2 alue om 20 oa samples om a
ood company was 0.71, which was also less han o
g ain samples g ound a MTT (Figu e 2).
The e was a good co ela ion be ween he DON
le els ob ained om 30 oa , 20 ba ley and 21 sp ing
whea samples using acc edi ed GC-MS and he
Rida®Quick me hod (Figu e 3). Pea son co ela-
ion alues we e 0.935, 0.931 and 0.935, espec i ely,
o oa , ba ley and sp ing whea samples. R2 alues
we e 0.88, 0.80 and 0.89, espec i ely, o he oa ,
ba ley and sp ing whea samples. Howe e , pai ed
- es s ( - alues 5.694, 7.854 and 3.544, espec i ely,
o he h ee g ain ypes) showed di e ences a he
95% signi icance le el be ween he Rida®Quick and
GC-MS esul s in all h ee ce eals. This was main-
ly due o he g ea e DON alues ob ained using
Rida®Quick me hod, including se en alse posi i e
oa and i e alse posi i e ba ley and whea samples
wi h Rida®Quick. 3ADON le els we e measu ed in
oa samples and hey we e usually abou 10–20% o
he DON le els.
The ep oducibili y o DON le els is shown in
Table 1. Comme cial whea s anda d (Rome Labs
1.431 mg kg-1) esul ed on a e age 2.11 mg kg-1 wi h
RSD 21 %.
When he DON le els om oa g ain samples
g ound a MTT and analyzed wi h he acc edi ed
GC-MS we e compa ed o hose ob ained om he
same samples in wo indus ial labo a o ies and
TUAS wi h he Rida®Quick me hod, he co ela ions
we e high (R2 usually g ea e han 0.89; Figu e 4), al-
hough DON amoun s ob ained wi h GC-MS we e
usually less in oa , whea and ba ley samples (Table
1). The co ela ion was also good, when DON le els
om whea g ain samples g ound in Labo a o y 2
wi h a mill ha ing a sie e we e compa ed wi h he
Rida®Quick me hod in he h ee labo a o ies (R2 >
Figu e 1. Co ela ion be ween log Fusa ium g aminea um
DNA and log deoxyni alenol (DON) concen a ion in
Finnish oa g ain samples in 2013. In all samples DON
was measu ed a MTT using acc edi ed GC-MS. DNA was
measu ed ei he om g ain samples g ound, a MTT, in a
mill wi h a sie e o om he same g ain samples g ound
wi h a mill wi hou a sie e, a UTU. Reg ession slopes and
R2 (coe icien o de e mina ion) alues a e shown. * and
*** indica e s a is ically signi ican (P<0.05 o 0.001).
Figu e 2. Co ela ion be ween log Fusa ium g aminea um
DNA and log deoxyni alenol (DON) concen a ions in
Finnish oa g ain samples in 2013. DON le el was es i-
ma ed using he RIDA® QUICK SCAN. Oa samples we e
g ound by a milling company o DON analysis, o by
Tu ku Uni e si y wi hou sie ing o DNA ex ac ion. Re-
g ession slopes and R2 (coe icien o de e mina ion) alues
a e shown. *** indica es s a is ically signi ican (P<0.001)
R2 = 0.71***
R2 = 0.76***
R2 = 0.31*
171
Vol. 56, No. 1, Ap il, 2017
G ain g inding and Fusa ium g aminea um de ec ion
Figu e 3. Co ela ions be ween log deoxyni alenol (DON) concen a ions in Finnish oa (a), ba ley (b) and whea (c) g ain
samples in 2013, es ima ed using he RIDA® QUICK SCAN compa ed wi h o log DON concen a ions ob ained a MTT
by using GC-MS. The samples we e g ound using he same mill a MTT o DON es ima ion, wi h sie ing. Reg ession
slopes and R2 (coe icien o de e mina ion) alues a e shown. *** indica es s a is ically signi ican (P<0.001).
Figu e 4. Co ela ions be ween log DON concen a ions in oa g ain p ocessed a wo milling company labo a o ies (Lab 1
and Lab 2), compa ed wi h DON concen a ions measu ed a TUAS and MTT. The g ain samples we e g ound a MTT (a)
in a mill wi h a sie e, o a Lab 1 (b) in a mill wi hou a sie e. *** and ** indica e s a is ically signi ican (P<0.001 o 0.01).

Phy opa hologia Medi e anea
172
T. Yli-Ma ila e al.
0.9, Figu e 5). In con as , when DON le els om oa
g ain samples g ound in Labo a o y 1 wi h a mill
wi hou a sie e we e compa ed wi h Rida®Quick
me hod in he h ee labo a o ies, he co ela ions
we e less co ela ed (usually < 0.8, Figu e 5).
Discussion
Ou esul s indica e ha he semiquan i a i e
Rida®Quick me hod o DON de ec ion in mos cas-
es me he equi emen s o he Commission Regula-
ion (EC) 401/2006 o DON de e mina ion, since he
RSD pe cen age alues we e equal o o less han 20.
DNA le els o F. g aminea um in oa g ains we e,
in all cases, in ag eemen wi h DON le els when
DON was measu ed by acc edi ed GC-MS. When
he Rida®Quick DON ki esul s we e compa ed
o DNA le els o F. g aminea um, he a ia ion was
much g ea e . Secu ing he homogeniza ion o he
oa lou using a 1 mm sie e as compa ed o g inding
wi hou a sie e was associa ed wi h he a ia ions o
DON and F. g aminea um DNA le els. These esul s
a e in ag eemen wi h p e ious esul s (Yli-Ma ila e
al., 2013; Hie aniemi e al., 2016).
Mo e alse posi i e esul s we e also ound in all
h ee ce eals when using he Rida®Quick me hod as
compa ed o acc edi ed GC-MS de ec ion. This was
due o he gene ally highe DON le els ob ained wi h
he Rida®Quick me hod. The alse posi i e esul s
may ha e been a leas pa ially due o 3ADON ound
de ec ed in oa samples. Excessi ely high alues o
DON ha e also been p e iously de ec ed using some
apid immunological es s (e.g. Aamo e al., 2012).
The DON le els ob ained wi h he Rida®Quick
me hod we e usually g ea e han hose ob ained
Figu e 5. Co ela ions be ween log DON concen a ions in whea g ain samples g ound a Lab 2 (a) and MTT (b), bo h in
mills wi h sie es. *** indica es s a is ically signi ican (P<0.001 o <0.01).
173
Vol. 56, No. 1, Ap il, 2017
G ain g inding and Fusa ium g aminea um de ec ion
wi h he acc edi ed GC-MS me hod, in all h ee ce e-
als. Howe e , he semiquan i a i e Rida®Quick DON
me hod used he e me , in mos cases, he equi e-
men s o he Commission egula ion (EC) 401/2006
o a quan i a i e DON de e mina ion. This should
show RSD % equal o less han 20. Bu wi h low DON
le els in ba ley and whea , he RSD % was abo e 20%.
Fo consume s, low DON amoun s gua an ee
ha he DON le els a e below he EU limi , bu o
a me s hey mean ha hei c ops may be ejec ed
due o alse posi i e DON alues. We conclude ha
bo h RIDA® QUICK SCAN and qPCR can be ap-
plied o sc eening o de ec ion o high DON le els
in ce eal g ain samples, bu he Rida®Quick assays
indica ing DON con en s close o he legisla i e lim-
i s should be epea ed and, i necessa y, econ i med
wi h an acc edi ed quan i a i e analysis. In u u e i
is impo an ha assay esul s ob ained om di e -
en apid immunoassay ki s used by ood indus ies
a e compa ed o hose ob ained using acc edi ed
chemical analyses.
Acknowledgemen s
This esea ch was inancially suppo ed by he
Tu ku Uni e si y Founda ion, he Ol i Founda ion,
he Egyp ian mission depa men , and a el g an s
o PhD s uden Taha Hussien o he Uni e si y o
Tu ku, om The Cen e o In e na ional Mobili y,
Finland (CIMO).
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Accep ed o publica ion: Feb ua y 28, 2017
Published online: May 10, 2017