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2008 The Authors

Journal compilation 2008 Blackwell Publishing Ltd


Molecular colog! "esources #200$% 9& '()'* doi+ (0,((((-.,(/**00$$8,2008,02(8',1
TECHNICAL AD VA NCES
An inexpensive high-throughput method to extract high
ie!ds o" good #ua!it DNA "rom "ungi
T, 2 3 2 4 5 6 3 & 7 , 2 A " 3 M & 6 , M A 8 4 9A & 9, : TA and T, 2 4 B : 7 :
Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 3058687, Japan
A$stract
%e deve!oped an e""icient method "or high-throughput extraction o" high-#ua!it DNA
"rom various "ungi& In this method' "unga! mce!ia (ere cu!tured and harvested on the
sur"aces o" mem$ranes on media p!ates& %e degraded ce!! (a!!s using a !tic en)me
*+ata!ase,& -uri"ication (as per"ormed on 9.-(e!! g!ass "i$re "i!ter p!ates& DNA (as
success"u!! extracted "rom various "ungi provided */01 genus /21 species, at high ie!ds
and #ua!it' and proved suita$!e "or storage' po!merase chain reaction amp!i"ication and
restriction en)me digestion& The method descri$ed is rapid' inexpensive and automation
"riend!& This ena$!es the simu!taneous extraction o" !arge num$ers o" samp!es'
signi"icant! improving the potentia! throughput in genomics' particu!ar! in diagnostic
and popu!ation studies&
ey!ords+ ;ilamentous ;ungi& genomic <7A& high0throughput method& P5"& 9atalase
Recei"ed #$ %ctober #007& re"ision accepted '3 February #008
Introduction
1traction o; genomic <7A ;rom ;ilamentous ;ungi is
labourious and time0consuming, =ilamentous ;ungi ha>e
e1tremel! hard cell walls and possess pol!saccharide and
pol!phenolic compounds& which are di;;icult to remo>e
and can strongl! inhibit reactions integral to <7A
anal!sis #Al08amarrai ? 8chmid 2000%,
5on>entional methods ;or <7A e1traction rel! on the
mechanical disruption o; m!celia using grinders #with or
without li@uid nitrogen% to reduce the plasticit! o; the
cell wall& and re@uire cet!ltrimeth!l ammonium bromide
#5TAB% and phenol)chloro;orm to remo>e pol!saccha0
ride and pol!phenolic compounds #>an Burik et a(, ($$8%,
These methods are time0consuming and cannot be auto0
mated& thereb! precluding their usage in high0throughput
pro.ects,
5hele10based protocols are a @uick and low0cost option&
but <7A e1tracted in this wa! is generall! low !ield and
unstable #Manian et a(, 200(%, Man! other rapid e1traction
methods ;or pol!merase chain reaction #P5"%
ampli;ication ha>e been reported #5enis ($$2A
6augland et a(, ($$$A Liu et a(, 2000A 2arakousis et a(,
200BA 8uCuki et a(, 200B%, Although these methods enable
@uick P5"0based anal!ses&
5orrespondence+ Taisei 2ikuchi& =a1+ 8( 2$ 8/D(*'DA 0mail+
kikuchitEa ;;rc,go,.p
2008 The Authors
Journal compilation 2008 Blackwell Publishing Ltd
the! are unsuitable ;or obtaining large amounts o; pure
<7A ;or anal!ses like 8outhern blots and ampli;ied
;ragment length pol!morphism #A=LP%& as well as ;or
storage,
8e>eral commercial <7A e1traction kits can success;ull!
be used with ;ungi #Fri;;in et a(, 2002%, Most o; these
products use silica0based methods and deli>er ;aster&
more stable and more sensiti>e <7A e1tracts, 6owe>er&
the cost o; these kits remains high and is o;ten prohibiti>e
;or large0scale anal!sis,
6ere& we present a new method utiliCing enC!matic
l!sis o; the cell wall and glass ;ibre0based puri;ication o;
<7A using $B0well plates, This method is rapid&
ine1pensi>e and automation ;riendl!& and can produce
high !ields o; good @ualit! <7A in a high0throughput
manner,
3ateria!s and methods
Rea)ents and supp(ies
The reagents are readil! a>ailable as standard
components& such as Tris& <TA& 7a5l& ethanol and 20
propanol& or as premade solutions& such as bu;;er AP2
#G3AF7%, He chose the AcroPrep $B =ilter Plate #Pall%
as our ;ibre ;ilter plate ;or binding genomic <7A, The
n!lon membrane used was Biod!ne A 8* mm disc
#Pall%, 5ellophane membrane was purchased at a
stationer& cut into s@uare pieces o; * I * cm& washed in
water and autocla>ed,
41 T5673 5AL A<JA 758
Ta$!e / =ungal species ;or <7A e1traction
Ascom!cetes *isce((a castanea +oni(ia kusanoi Rhi,osphaera sp,
-.phiporthe ra"ene(iana *othidea sp, +onochaetia .onochaeta /area resinae
-p(ospore((a sp, *othiore((a sp, +onostiche((a sp, /c(erotinia sc(erotioru.
-rthriniu. phaeosper.u. *repanope,i,a puncti0or.is +ycosphaere((a (u,onensis /e(enopho.a sp,
-scochyta pisi 1ndothia radica(is +ycosphaere((a .yricae /eptotis sp,
-sperisporiu. se2uoiae 1ndothie((a sp, +ycosphaere((a to)ashiana /phaeropsis crypto.eriae
3arta(inia robi((ardoides 1nto.osporiu. sp, +y4osporiu. rhois /phaeropsis sapinea
3otryosphaeria dothidea 1picoccu. ni)ru. +y4osporiu. sp, /ta)onospora cinna.o.u.
3otrytis cinerea 1picoccu. sp, 5ectria cinnabarina /ta)onospora sp,
6a(onectria kyotensis Fusariu. o4ysporu. 5ectria sp, /trasseriopsis tsu)ae
6enan)iu. 0erru)inosu. Fusariu. 0u7ikuroi 5eocos.ospora sp, Taphrina !iesneri
6enan)iu. sp, 8ibbere((a baccata %phiosto.a piceae Trichotheciu. roseu.
6eratocystis .inor 8(ioc(adiu. sp, %phiosto.a sp, Trichoder.a (on)ibrachiatu.
6ercospora popu(ico(a 8(oeosporiu. ka!aka.ii Pate((ina sp, Trochophora 0ascicu(ata
6ercospore((a 7u)(andis 8(oeosporiu. sp, Penici((iu. citrinu. Truncate((a sp,
6haeto.iu. 0unico(a 8no.onia setacea Penici((iu. indonesiae Tubercu(ispora sp,
6haeto.iu. )(obosu. 8ui)nardia a(ni)ena Penici((iu. .e)asporu. 9a(sa kun,ei
6haeto.iu. sp, 8ui)nardia (aricina Penici((iu. pseudostro.aticu. 9a(sa ni"ea
6(adosporiu. sp, :a(bania 7uniperi Penici((iu. "errucosu. 9a(saria sp,
6o((etotrichu. coccodes :endersonia sp, Periconia a,a(eae 9ertici((iu. sp,
6o((etotrichu. truncatu. ;achne((u(a resinaria Pesta(otia aceris <ythia sp,
6orynespora cassiico(a ;epteutypa cupressi Pesta(otia popu(i=ni)rae
6oryneu. sp, ;epto)raphiu. (undber)ii Pesta(otiopsis )(andico(a Basidiom!cetes
6ryphonectria parasitica ;epto)raphiu. proceru. Pesta(otiopsis )uepini -.y(ostereu. areo(atu.
6ryptodiaporthe castanea ;epto)raphiu. !in)0ie(dii Phaeoseptoria euca(ypti 14obasidiu. bisporu.
6y(indrosporiu. sp, ;eptosphaeria sp, Phia(ophora sp, F(a..u(ina "e(utipes
*iaporthe .edusaea ;eucosto.a persoonii Pho.a sp, :e(icobasidiu. .o.pa
*iaporthopsis sp, ;ophoder.iu. pinastri Pho.opsis rudis :e(otiu. (euce((u.
*iatrype sp, +acropho.a 2uercico(a Phy((osticta a(cides +undkure((a ka(opanacis
*iatrype((a sp, +acropho.a su)i P(ectosphaera crypto.eriae Thanatephorus cucu.eris
*icarpe((a dryina +acropho.ina phaseo(ina Racodiu. therryanu.
*idy.e((a sp, +e(anconis 7u)(andis Ra00ae(ea 2uerci"ora
*ip(ocarpon .espi(i +e(anconis sti(bosto.a Retinocyc(us sp,
*ip(odia sp, +e(anconiu. ob(on)u. Rhi,ina undu(ata
*ip(odina popu(i +etasphaeria sp, Rhi,osphaera ka(kho00ii
The 9atalase l!sis solution was composed o; (K
9atalase #Ta2a"a%& 200 Lg-mL "7ase A #3n>itrogen%& (0
mm sodium phosphate #p6 B%& 0,8 M 7a5l& and *0 mm
<TA #p6 /%& and was steriliCed b! ;iltration be;ore use,
The 8<8 l!sis solution was composed o; 8K 8<8&
0,* mg-mL proteinase 2& (00 mm 7a5l& (0 mm Tris #p6
8%& and ( mm <TA,
/a.p(e
preparation
The ;ungal strains used in this stud! were ;rom the
culture collection stored within the =orest Patholog!
Laborator! at the =orestr! and =orest Products
"esearch 3nstitute o; Japan, =ungi were cultured on
cellophane membranes placed on potato de1trose agar
#P<A& iken 5hemical% plates at 2D M5 ;or periods
appropriate ;or each ;ungus, =ungal m!celium was
har>ested ;rom each plate into (,*0mL tubes or the wells o;
$B0deep0well plates b! scratching the sur;ace o; the
membranes using steriliCed small metal spatulas, 3n
each case& the m!celium was stored at )80 M5
T5673 5AL A<JA 758 42
;or ;urther <7A e1traction, He used n!lon membranes
in place o; cellophane membranes onl! when it was
di;;icult to har>est m!celium because o; se>ere
degradation o; cellophane membranes during ;ungal
culti>ation,
To test this method& we used m!celial samples #()D0
mg% o; a >ariet! o; ;ungi ;or <7A e1traction& including
(2* species o; ascom!cetes and / species o;
basidiom!cetes #Table (%,
*5-
puri0ication
<7A puri;ication was per;ormed as
;ollows+
/ 9atalase l!sis solution #200 LL% was added to each
tube or well and incubated at D/ M5 ;or 0,*)( h with
agitation at (000 r,p,m, using a shaking incubator
#32(00J& 9amato 8cienti;ic 5o%,
1 8<8 l!sis solution #B/ LL% and (00 LL o; Circonia
beads #0,* mm diameter% were added to each tube
or well& >orte1ed ;or ( min and incubated at B0 M5 ;or
(0 min,
2 8olution AP2 #8/ LL% was added to each tube or well,
A;ter incubation on ice ;or * min& samples were
centri;uged at
(* 000 g ;or * min #(,*0mL tubes% or D000 g ;or (* min
#$B0 deep0well plates% to pellet the debris,
4 Be;ore trans;erring ('0 LL o; the cleared l!sate to a $B0
well glass ;ibre ;ilter plate& 220 LL o; 20propanol was
dispensed into each tube o; the plate,
5 A;ter a static *0min wait to ;acilitate binding o; the 20
propanol0precipitated <7A to the glass ;ibres& >acuum
pressure was applied using a Millipore >acuum mani0
;old to remo>e the cell l!sate solution,
. The glass ;ibre ;ilters were then washed ;our times with
200 LL o; 80K a@ueous ethanol b! >acuum ;iltration,
6 =ilter plates were centri;uged at D000 g ;or D min to
remo>e residual ethanol and dried using a >acuum
e>aporator,
7 Plates were resuspended with B0 LL o; preheated #B*
M5% T #(0 mm Tris)65l& p6 8,0& 0,( mm <TA%&
allowed to stand ;or * min and then eluted b!
centri;ugation at D000 g ;or * min into a $B0well
collection plate, This elution step was repeated once&
resulting in (20 LL o; ;inal elution >olume,
P6R a.p(i0ication
The large subunit region o; ribosomal <7A was ampli;ied
using P5" primers nu0L84000'2)*N #L"0"% #A555F5TFA0
A5TTAAF5% and nu0L8400$'8)DN #L"*% #T55TFAFFF0
AAA5TT5F% #a location map and oligonucleotide se@uences
o; these primers can be ;ound at ww w ,biolog ! ,duke,edu-
;ungi-m!colab-primers,htm%, P5" ampli;ications were
carried out in D00LL reaction mi1tures containing (* LL
FoTa2 Freen Master Mi1 #Promega%& 0,* Lm o; each primer
and ( LL o; each <7A e1tract, 5!cling conditions were
$' M5 ;or ( min& then D0 c!cles o; $' M5 ;or D0 s& *D M5 ;or
D0 s and /2 M5 ;or ( min,
*i)estion !ith restriction en,y.es
"estriction enC!me digestion was per;ormed in D00LL
reaction mi1tures containing the appropriate bu;;er #(I%&
20
4 o; each restriction enC!me #3a.63& 1co"3& :ind333 or
Pst3% and (0 LL o; each <7A e1tract, To assess the auto0
degradation o; <7A& a <7A sample was also incubated
under the same conditions but in absence o; restriction
enC!me,
8esu!ts and discussion
Hith this method& <7A was success;ull! e1tracted ;rom
>arious ;ungi e1amined #(D2 species% #Table (%, <7A
@ualit! was tested b! direct electrophoresis, 6igh
molecular weight <7A was obser>ed when * LL o; the
<7A solution was electrophoresed on (K agarose gel and
>isualiCed b! ethidium bromide staining #=ig, (%,
9ig& / Agarose gel #(K% electrophoresis o; e1tracted <7As ;rom
(B species o; ;ungi, =i>e microlitres o; e1tracted solutions was
loaded, M& 20log ladder #0,* Lg%A (& 6eratocystis .inorA 2&
;epto)raphiu. !in)0ie(diiA D& +onochaetia .onochaetaA '& ;epteutypa
cupressiA *& +ycosphaere((a to)ashianaA B& +ycosphaere((a (u,onensisA /&
%phiosto.a piceaeA 8& %phiosto.a sp,A $& Pesta(otiopsis )uepiniA (0&
Pesta(otiopsis )(andico(aA ((& Phaeoseptoria euca(yptiA (2& Rhi,ina
undu(ataA (D& Retinocyc(us sp,A ('& /area resinaeA (*& Penici((iu.
indonesiaeA (B& Penici((iu. pseudostro.aticu.,
9ig& 1 P5" ampli;ication o; ribosomal <7A o; (B species o; ;ungi,
The large subunit o; ribosomal <7A was ampli;ied and * LL o;
P5" products was loaded on a (K agarose gel, 7umbers
correspond to the species listed in =ig, (,
<7A concentrations were (0)(00 ng-LL #appro1imatel!
(,2)(2 Lg o; <7A in total elution% and 2B0-280 ratios
were
(,B)(,$ when measured b! 7ano<rop spectrophotometr!
#7ano<rop Technologies%, 3n the case o; one
representati>e species& Ra00ae(ea 2uerci"ora& we obtained
appro1imatel! ( Lg o; <7A ;rom (0 mg o; m!celium
sample with this method, This amount was comparati>el!
high& indicating the pre0 sented method is e;;icient ;or
obtaining pure <7A ;rom ;ungi, Moreo>er& <7A e1tracts
were used as templates ;or P5" ampli;ication o; the
large subunit region o; ribosomal <7A, An
appro1imatel! $000bp ;ragment was success;ull!
ampli;ied ;rom each <7A e1traction #=ig, 2%,
=urthermore& <7A e1tractions were e;;icientl! digested
with restriction enC!mes #=ig, D%& while no degradation
was obser>ed in e1tracted <7A incubated under the same
condition without restriction enC!mes #data not shown%&
impl!ing that the @ualit! o; the <7A e1tracted b! this
method was su;;icient ;or digestion b! restriction
enC!mes, He suggest that <7A puri;ied b! the presented
method is o; such high @ualit!
9ig& 2 <igesti>it! o; <7A prepared with the presented method,
Ten microlitres o; e1tracted <7A was digested with 3a.63 at
D/ M5 ;or D h in D00LL reaction mi1ture and B LL o; the mi1ture
was loaded on a (K agarose gel, 7umbers correspond to the
species listed in =ig, (, 8imilar electrophoresis patterns were
obtained when <7A was digested with 1co"3& :ind333 or Pst3 >not
shown%,
and !ield that it can be used not onl! ;or P5" ampli;ication
but also ;or anal!ses that re@uire large amounts o; high
@ualit! <7A& such as 8outhern blot or A=LP anal!sis,
3n addition& <7A @ualit! a;ter storage at ' M5 ;or
B months did not diminish& measured b! electrophoresis
images and the le>el o; P5" ampli;ication #data not shown%,
He belie>e @ualit! o; entire set o; <7A will remain intact
a;ter storage ;or more longer period o; time,
3n this stud!& we demonstrated a new method ;or high0
!ield& high0@ualit! <7A e1traction ;rom >arious ;ungi,
This e1traction and puri;ication procedure is likel! to be
applicable ;or broad ranges o; conditions& including a
wide breadth o; the 2ingdom =ungi, Hith this method&
<7A was e1tracted ;rom >arious ;ungal tissues including
tissues that ha>e been preser>ed in absolute ethanol&
spores and ;resh& ;roCen or dried ;ruit bodies, <7A
were readil! e1tracted ;rom all samples and
electrophoresis images o; e1tracted <7A were similar to
those o; <7A e1tracted using commercial kits #<7eas!
Plant mini kit& G3AF7% #data not shown%,
3t is widel! accepted that some ;ungi ha>e highl! acti>e
nucleases& which pre>ent e1traction o; high molecular
weight <7A, 3n con>entional methods& harm;ul protein
denaturants such as phenol or guanidine thioc!anate are
used to inacti0 >ate the nucleases, Because we did not use
these protein denaturants in this method& degradation o;
<7A during the e1traction process ma! ha>e occurred,
6owe>er& we obtained high molecular weight <7A ;rom
all ;ungi used, <7A degradation might be minimiCed
b! handling the ;roCen samples @uickl! so that the!
cannot be dissol>ed be;ore addition o; l!sis bu;;er
containing *0 mm <TA,
Flass substrates #particles& beads and ;ibres% ha>e been
used ;or puri;!ing nucleic acids under >arious conditions&
including <7A in agarose gels #Jogelstein ? Fillespie
($/$%& plasmid <7A in bacteria #<e derich et a(, 2002% and
genomic <7A in animal or plant tissues #3>ano>a et a(,
200B%, Binding o; <7A to glass substrates is generall!
;acilitated
b! the presence o; high concentrations o; chaotropic salt
such as sodium iodide and sodium perchlorate #Jogelstein
? Fillespie ($/$A 6oarau et a(, 200/% or b! the presence
o; high concentrations o; sodium chloride and
pol!eth!lene gl!col #PF% #ngelstein et a(, ($$8%, These
methods ha>e the common element o; high salt solutions&
in which the adsorption o; plasmid <7A onto the glass
substrate occurs most likel! b! a mechanism similar to
adsorption chroma0 tograph! #<e derich et a(, 2002%,
6owe>er& unlike the high salt methods& we used 20
propanol to precipitate the <7A onto glass sur;ace, This
method has been success;ull! used to puri;! plasmid
<7A ;rom 1scherichia co(i #<e derich et a(, 2002%, 3n this
stud!& we showed the method can be used ;or genomic
<7A preparation ;rom ;ungi& as well,
3n ;act& our newl! de>eloped method o;;ers some
de;inite ad>antages o>er traditional methods o;
;ungal <7A e1traction, =irst& m!celium used ;or <7A
e1traction was collected ;rom the sur;aces o; cellophane
or n!lon mem0 branes on P<A plates, 3n con>entional
methods& li@uid culture was ;re@uentl! used to grow
;ungi be;ore <7A e1traction, 3n this method& b!
culturing on membranes on P<A plates& we reduced the
risk o; media contamination& which sometimes inhibits
subse@uent enC!matic reactions, Additionall!& the risk o;
bacterial or ;ungal contamination can also be minimiCed
because the colonial morpholog! o; each ;ungus can be
>isuall! checked on the plate be;ore collection, 8econd& a
l!tic enC!me #9atalase% was used to destro! cell walls o;
the ;ungi, This replaced the traditional mechanical
disruption step& which is labourious due to the
di;;icult! in disruption o; ;ungal cell walls b! brie;
mechanical methods such as bead mills or sonication&
which also incurs signi;icant losses when mortars and
pestles are used ;or disruption, nC!matic l!sis is non0
laborious and enables the procedure to be completed in
one tube, =inall!& the e1traction and puri;ication steps
were accomplished with $B0well plates& which reduce the
labourious handling o; multiple samples& are automation
;riendl! and allows ;or e;;icient downstream processing,
Thus& our de>eloped method achie>es great sa>ings in
both time and cost, The e1traction o; $B samples ;rom
m!celia in tubes-wells can be completed within D h, The
appro1imate cost o; each <7A e1tract prepared b! the
present method is O P0,** making it si1;old cheaper
than commerciall! a>ailable kits,
The o>erall picture emerging ;rom this stud! is the
de>elopment o; rapid& ine1pensi>e and reliable method
;or the e1traction o; genomic <7A ;rom ;ungi, To
understand the ;ungal genetics and their ecological
relationship& it is necessar! to obtain a high !ield o;
;ungal <7A with good @ualit!, The high0throughput
e1traction method designed and de>eloped in this stud!
will pro>ide a better opportunit! ;or complete and precise
anal!sis o; ;ungal genomes in a wa! to understand the
molecular ecolog! o; >arious kinds o; ;ungi,
Ac:no(!edgements
The authors thank Asuka 8hichiri ;or technical assistance, This
work was ;unded b! the Japanese Ministr! o; ducation& 8cience&
8ports and 5ulture& Frant0in0Aid ;or ncouragement o; 9oung
8cientists #B% (8/800D2 #T2%& (///00/* #9:% and Frant0in0Aid ;or
1plorator! "esearch (8B*/0D2 #6M%, 72 is supported b! Japan
8ociet! ;or the Promotion o; 8cience #J8P8% Postdoctoral
=ellowship ;or ;oreign researchers,
8e"erences
Al08amarrai T6& 8chmid J #2000% A simple method ;or e1traction
o; ;ungal genomic <7A, ;etters in -pp(ied +icrobio(o)y& 20&
*D)*B,
>an Burik JA& 8chreckhise "H& Hhite T5& Bowden "A& M!erson <
#($$8% 5omparison o; si1 e1traction techni@ues ;or isolation o;
<7A ;rom ;ilamentous ;ungi, +edica( +yco(o)y& 2.& 2$$)D0D,
5enis JL #($$2% "apid e1traction o; ;ungal <7A ;or P5"
ampli;ication,
5uc(eic -cids Research& 10& 2D80,
<e derich <A& :kwuonu F& Farner T et a(, #2002% Flass bead
puri;i0 cation o; plasmid template <7A ;or high throughput
se@uencing o; mammalian genomes, 5uc(eic -cids Research& 20&
eD2,
ngelstein M& Aldredge TJ& Madan < et a(, #($$8% An e;;icient&
automatable template preparation ;or high throughput
se@uencing, +icrobia( and 6o.parati"e 8eno.ics& 2& 2D/)2'(,
Fri;;in <H& 2ellogg 5A& Peak 22& 8hinn A #2002% A rapid and
e;;icient assa! ;or e1tracting <7A ;rom ;ungi, ;etters in -pp(ied
+icrobio(o)y& 24& 2(0)2(',
6augland "A& 6eckman JL& H!mer LJ #($$$% >aluation o;
di;;erent methods ;or the e1traction o; <7A ;rom ;ungal
conidia b! @uantitati>e competiti>e P5" anal!sis, Journa( o0
+icrobio(o)ica( +ethods& 26& (B*)(/B,
6oarau F& 5o!er JA& 8tam HT& :lsen JL #200/% A ;ast and
ine1pen0 si>e <7A e1traction-puri;ication protocol ;or brown
macroalgae, +o(ecu(ar 1co(o)y 5otes& 6& ($()($D,
3>ano>a 7J& <ewaard J"& 6ebert P<7 #200B% An ine1pensi>e&
automation0;riendl! protocol ;or reco>ering high0@ualit! <7A,
+o(ecu(ar 1co(o)y 5otes& .& $$8)(002,
2arakousis A& Tan L& llis <& Ale1iou 6& Hormald PJ #200B% An
assessment o; the e;;icienc! o; ;ungal <7A e1traction methods
;or ma1imiCing the detection o; medicall! important ;ungi
using P5", Journa( o0 +icrobio(o)ica( +ethods& .5& D8)'8,
Liu <& 5oloe 8& Baird "& Pederson J #2000% "apid mini0
preparation o; ;ungal <7A ;or P5", Journa( o0 6(inica(
+icrobio(o)y& 27& '/(, Manian 8& 8reeni>asaprasad 8& Mills P"
#200(% <7A e1traction method ;or P5" in m!corrhiCal ;ungi,
;etters in -pp(ied +icro=
bio(o)y& 22& D0/)D(0,
8uCuki 8& Taketani 6& 2usumoto 2& 2ashiwagi 9 #200B% 6igh0
throughput genot!ping o; ;ilamentous ;ungus -sper)i((us
ory,ae based on colon! direct pol!merase chain reaction,
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