Development of simple molecular protocols to detect Banana bunchy top virus with the phytoPaSS System

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Proc lS on Banana Crop p1s1 ' lust

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& lnrPr' Lir clihoods

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Despite

its

high

sensitivity,

pcR

is not

used conrmonly,

o'

a

large scare. A contribtrtirrg

_hctor

rlrl

_be

the,need^lor

.purilieci ntrcleic

u.iJi

ur

remptàr.

*.ilri.i

(Furuya

et

al..

200,s;

su

et

al.,

200j;

ivanitchakorn

.t

oL.,

-

zôooi. irr".r.i.

--à.;î,

purification from banana tissLres is time consumin-q

and.un

t.ià

lo"miniputatio;.;;;;;

lhcn

higlr nurnbers of samplcs arc anrlrsed.

this paper.

ue

inrestiglte

a

iirlplified^protocol

for the molecular cletection

_of

BBTV that does nor use pLrrified-DN,A

prioi

ro

pcit

amplification.

ùIATERIALS

_A\D

NIETHODS

Plant

llaterials

and Preparation of Banana Samples

for

BBTV Detection

BBTV-inlected banana plants fronr the Democratic Republic

of

Con-co and iiorn

_BrrrLrntii \\erc^s,ro\\n

in

the gieenhouse under

a

constant teirperrture

oii,+.c

aftl;

phoropertotl

ot l6

hoLrrs.per. day'. Leaves

ol

each banana plant^*,ere scrLrbbed

",itf-1 inJ

PhltoPASS

_tD\Alis

sprl)

abiaiire

mernbrane

to

derach prort

ti.ii.r

_ï..oraing

_to

_tii.

Phr'toPASS rnantrtàcttrier;s protocol. (Frg.

it.

The nrernbrane coverecl u:irh fragnients

_of

banana tissues

uas

then inirodr-rceci'inù

_a

15-ml tLrbe

containing-l

".lf

ol

à;a

ilji

extraction bufler

(DN,Al'::qn,,

and the preparation i.ias vorrered-at rri-un

speeJlilbî-2000 rpm tbr 30 sec) to

_lield

the

prinrarljcrrde

ertract of banana.

a

-ei.Jtr sr-rspension

ol

plant extracts \\'as thus generatecl _{âs shorin on Figure}

_l.

-Consen'ation of Plant 'Iissues on the phl.topASS _Surface

Sarnples, ha^'ested frorn a banano plant

fr.rn

the Dernocratic Republic or conso

\\ere

conser\ed

on

thc

PhvtoPASS abrasive meurbranc

to e\ûlutrc

tire leasiUitity

ii

consening

the _.sarnples

bitbre

molecular anall'sis.

1n. r.rrlpii'i"ti.r.'r..pt

at

roour

l:i,]p:i.!,1r"

on the PhltoPASS inside paper e'relopes for 32.

qô,lz

and r38'days. After

thls stora-ge _{period, tlre samples \\'ere recovered anddilutecl in the above-mùntion;d KAJI}

ertraction bufÈr prior to PCR.

Nlolecular Detection of

BBTV

The prinrar'"_extract rl'as dilirted 100 times in sterile distilled n.ater and conserved

b)

Ïr_eezrns

_rt

-10'C

fbr ftrture use as template

for

PCR amplification. pCR cjetection

of

BBTV

rvas underraken

riith

the

priner

pairs

(BBTI

&

BBj'zl

(inàn-'ro,iand

Dietzgen.

1995).

A

PTC 200 therrnocycler'(Biozym, _{Land-eraaf, thc NetÉerlanarj}

_ri:.r

_{used for the}

PCR amplification.

in

a

flnal

rolume

ôt

so

,ul iontaining

5 Fl

;itli.'r0.,

concentrarecl

,tjlI:l.y:]"

buûèr rRoch,er.200 trr\t oteach

d\Tp.0.5lL\l

oi'erctr prirrer and 5 prt or'

thc l00r

dtltrtcd

plrnt

crLrde

e\trtct

suspcnsirrrr. Anrplificririon eL,ndirion: inclrrclc6

I

Ilrst

denaturation srep ar

94'c

tbr 5

min

foiloued

b1 35 cycles

oll0

_s;.-;i9-+.c,

_I

min at

52"C and 2 min at 72'C. These repetitire.c,r,cles _{nere ftllolr,ed b1.a final elongation step} pcIlbrmed at

7l

C

lor l0

nrin. PCR prodrr.'ts

\\cre

seprrated br-clectroplloresis rrndcr a

constrnl electric cLtrrenl

ol

120

mA

in

a

loo asaroqc gcl contrininu ethiàrLrm bronridc _1l

pLg/10 ml). The amplificd prodLrcts rrere visuelÈed und"er UV liqht.

RESULTS

A\D

DISCUSSIO\

Reco'ery of

crude Ertracts

anrl

Efficicnq

of

pcR

Detection of

BBT\'

,

The

prepar:ation

of

the phnt

ertrict

suspension r.las rapicl ancl

silrple.

The cqmplete process from samplin-e

io

the preparation olcmcle extracis

r.q,iii.A

leis than 5

m1n.

.

Figtrre

2

shorvs

the,amplilication

_{patterns obtainecl}

_for

_the

_tuo

_{banlna plants} subrnitted

to

this^analysis

ln

Lroth _{cuser, àn arnplifiecl product was obiained,}

_rviih

_the

crpcctcd size

ol3l9

bp {Thonrson irnd Dir'rzeen. I9o5

j

conllrmirrg rlLrr rhe plcnrs arc

intècted

l

ith BBTV.

The presenr assry shori,ed

lbr

the

first

time

inlections

b1

PCR

u

itli

crtrcie extracts preparecl

214

that

it

is

possible

to

detecr BBTV

from

samples collected

lvith

the

PhytoPASS.system. The prorocol siurplifies _{and speeds up the preparatory steps needed}

before actual PCR reactions can be perfornred.

^---.

Classical plotocois-require nucleic acid purification before PCR amplitication

lor

.BBTV derecriorr. The purificaiion of nucleic acibs in

ru,",.qriË.

nolnài.,,lrurlo,r of

lerf

tlssues ll'l an extraction buller and iltcubation of _{the preparati'on at high tErnperarure lor at}

least

th

_{(Su et al., 2003). optirnised protocols for nùcleic aciai purifrcaiiàn}

stitt require a

prelinrinary grinding step

oi

banana leaves

in

tiqura nitrogen

_{;Jf";;';;ii;r.nt}

_incubation

ïj,î:]Tr:},cîrron

sreps

jn

errracrior butfers tFLirupg err1..2005).

rt,.iè

oitr.r.nr

ri.pi

are tllne

alld

labour consunting.and inct'ease tJre risk

of

nranipulatioq errors

rs riell

:rs cross-cotttamination betueen dif-ferent sanrples. An altemative

_ô

irr.Ëi.

u.ia

pu.in.ui;à,i

u'as optinrised ro derect differenr balana viruses,_includiirg

gÈrv

_fji,r.;n

_{et al., 2000).}

The proposed reclrnology. irrr orves _{ilregrarion of} un.

i'r,ini,ro;;p,r.;;

_iiëj

srep precedins

the PCR reaction

to

aclrieve irnnrobiliiarion

of

vrnons orr the pCR reactiorr rube wall. Although-r'irus inimobilisation o\rercomes linritations caui.a 6v

Êèn i"rTtitors

in banana

extracts, the procedure requires. _{specific antibodies and reniains'titr.}

_co,ri*ning

.

The protocol described here significantly

sinplifies

a.t..ti""lîggrv

_{in infected} banana samples by using cr.ude e.rtracis for pCÉ a,nptin.utiÀn.

\/irus

Detection

in

Conserved Samples

^

..fht^aniplification

profiles

obtained

for

banana sanrples

fiorr

the

Derrocratic

Lt-r:llr":l.a:,1c_:]Tl:.1.d

*irrr

rrre phvopASS_svr,.','

u'iJ',ià,.Jài'oà,r'

rernperarure

ror

olllerent

storage.perrods

_{are illustrrted}

_in

_Figure

_3.

_BBTV

_{infections renrairred}

detectable lor at reast 4 rnonths (the longest storage périod tested) before

pcR

_analysis.

CONCLUSION

.

This.

simplified

detection _{protocol could be useful}

_in

_{rhe manasement}

_ol

_this important disease.

It

was shori'n ttràt

eerv

infections

are.^"iy'â.,.ir"ur!îiirriË'pëii

technology using clude exrracs as rarger nrareriars.

Tl*

;.;;i"ËJpài"."r

_overcomes

tlre need

ro.purifli

rrucleic acids

or ro

inresrare u,r

iir.,,uuïo-i;ôrr-".";;;

prior

ro

Rôii deteclion. \4oreover.

it

allous lire

corrservilion

of

sarnples under cornriron condirions. ertabling easy lransporl

_ol

_{sarrples and rhus bypassing}

_irr.

"..à-io-.iiulritrr

rrrolecular.

laDoratory capacllles tn a gtrett _{producing regiorr. Tlrese results could contr.ibute ro t5e}

developnrent of a global banarra healtir rnaiagôrént programrre.

A C KÀT O

\\'L

E D G E ]\{EN TS

The autlrors rvould like^to thank _{Angelo Locicelo and Laetitia Masquellier for} perlorrning tlte rnolecular anrpl i fi carion analvies.

Literature

Cited

Bananei,

K.,

Ghotbi.

T.

and Vahdat,

A.2007.

Fir-st report

of

Banana bunchy

lop'irus

^

infecting_banana in lran. plant pathology 56:7j9.

Beetharr, P.R., Harding, R.rv. and Dale,

iL.

r999. Banana bunchy top virus DNA-2 to 6

are ntonocistronic. Archir,es of Virolo-ey 144: g9_105.

Furuya, N.,. Kawano, S. and Natsuaki,

È.r.

zoo:.

characterization

of

genetic status

of

B,a'ana bunchy rop virus isolated fi'orn okinau,a, Japan. J. Gen plant Furhotogy

ir,og-I J.

Karan,

M., Hading,

RM

a1{

Dale,

l.L.

1994. Evidence

for

trvo

groups

of

banana

-

bunchy top virus isolates. J Gen

Virol.

75:3541_3546

' -

è'""

Sharrnan,

M.,

Thomas,

J.E. and

Dietzen,

R.G. 2000.

De'eropment

of a

rrultiprex

llllmunocapture PCR

u'ith

colourinretric detection

lor

viruses

of

banana. Joumal

of

Virological lr4ethods 89:75-8g.

Su,^H.J., Tsao,

L.Y.'

wu,

N{.L. and Hung,

T.H.2003.

Biological and

Molecurar

categorizatior and Strains

of

Banara burichy top virus.

Jounril

or

Ëhyopatt,ology 151:290-296.

Tho'rson, _{D. and Dietzgen, R.G. 1995. Detection of DNA and RNA}

plant viruses by

pcR

,1:!, "a :ji i :rl?l: ':â.: 215

.:

if

Fig' 2. i\'{olecuiar

detectio'

profile

of

BBTV

in

_lqnqqu- tissue suspensions pr-epared

according.to the

_l-2:

phyopASS protocol.

rane

M:

r00-bp-iadder

_{iÈ...',.ntur);}

tun.,

samples collected rrorn

tle

pranr fr-om Burundi;

l;,r;j-+,'r;ipres

collected from the planr from rhe Demociaric Repubric

"iôàiùi"r.,,e

il'jirtittea

,"ut.r,

negative control.

:l l l

3'

Detection of

BBTV

_{in banana sanrples fi.orr an infected plant that *,ere pr.ocessed}

)::,1^fr:"llli:^lASSsysten

and cônserved at roorr rernperarure. Lane Nt: 100 bp

la00er (,f _{ennentas): lanes I-3: sarnples lrcslrly collecred:}

lanes J-6: srrnples srored

Ior

i-:

days: lanes /-q: sarnples stor.ed lor .10 da1 s: lanes l0- I

l:

sarrrples srorcd lor

72days; lanes l3-15: sampies stored

for

l3g ciavs.