A reliable IC One-step RT-PCR method for the detection of BBrMV to ensure safe exchange of Musa...

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Journal of Virological Methods 153 (2008) 223–231

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Journal of Virological Methods

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reliable IC One-step RT-PCR method for the detection of BBrMVo ensure safe exchange of Musa germplasm

arie-line Iskra-Caruana ∗, Serge Galzi, Nathalie LaboureauIRAD-BIOS UMR 385 BGPI, Campus International de Baillarguet, TA A54/K, F-34398 Montpellier Cedex 5, France

rticle history:eceived 27 January 2008eceived in revised form 23 June 2008ccepted 25 June 2008

a b s t r a c t

An immunocapture (IC) One-step RT-PCR assay was developed to improve the detection of Banana bractmosaic virus (BBrMV) in single and bulked samples of banana plants. In this paper, an atypical strain ofBBrMV was described, the BBrMV “Ref” strain, and we showed that detection with available BBrMV toolsusing ELISA and RT-PCR approaches was not reliable. Primer sets Bract N1/NR and N2/NR specific to BBrMV

vailable online 20 August 2008

eywords:anana bract mosaic virus

C One-step RT-PCRLISA

were designed and used in RT-PCR and IC-RT-PCR assays with two commercial kits that allow the RT andthe PCR reactions to take place simultaneously in the same tube. The new assay enabled detection ofBBrMV in leaf extract diluted up to 1 × 10−10 and in bulked samples of 10 plants, and was proposed as anew international standard to index BBrMV.

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usa germplasmulked detection

. Introduction

Viruses have had – and continue to have – a major effect onany crops including banana, worldwide. At least five viruses have

een characterized in banana, and four of them are responsibleor diseases. However, the impact of each banana virus differsignificantly: Banana bunchy top virus (BBTV) remains the most eco-omically important virus in banana (Thomas and Iskra-Caruana,000), while Banana bract mosaic virus (BBrMV) is a local problem

imited to few countries (Thomas et al., 2000a), and, on its own,anana mild mosaic virus (BanMMV) has never been reported toause a disease even though it occurs widely in Musa germplasmThomas et al., 2000b). Usually banana plants are infertile andre propagated vegetatively. Traditionally, this has been done byuckers, but to facilitate the safe international exchanges, manyperators now use tissue culture. Banana viruses are transmittedy all vegetative planting material (suckers, bits and corms), andicropropagated plantlets facilitate large-scale dissemination of

iruses when infected material is multiplied. Sensitive and early

∗ Corresponding author. Tel.: +33 499624813; fax: +33 499624808.E-mail addresses: marie-line.caruana@cirad.fr (M.-l. Iskra-Caruana),

erge.galzi@cirad.fr (S. Galzi), nathalie.laboureau@cirad.fr (N. Laboureau).

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istribution, its ability to spread easily via the use of vegetativelant parts, aphid vectors, and its inconsistent production of vis-

ble symptoms (Magnaye and Espino, 1990; Thomas et al., 1997).nfected BBrMV plants can be either symptomless or display symp-oms that are more typical of chlorosis than of banana bract mosaicntil the appearance of the typical final symptom of the disease:mosaic of the flower bracts (Rodoni et al., 1999; Thomas et al.,

000a). BBrMV is reported to cause yield losses of up 40% in “Card-ba” (AAB) and “Lakatan” (AAA) cultivars in the Philippines and haseen extremely difficult to control (Kenyon et al., 1996; Thomasnd Magnaye, 1996). No effective resistance is known and the usef indexed virus-free planting material remains the best way torevent and to control the disease.

BBrMV is a member of the genus Potyvirus in the family Potyviri-ae and has a positive-sense ssRNA genome of approximatively0 kb (Gambley and Thomas, 2001; Rodoni et al., 1997; Thomast al., 1997). Serological and molecular detection assays have beeneveloped (Thomas et al., 1997) and Rodoni et al. (1999) showedhat RT-PCR was the most reliable assay for the detection of infectedanana plants even though it is known that the high sensitivityf RT-PCR can make it difficult to avoid contamination. However,his technique requires an RNA extraction step which lengthenshe sample preparation stage, and can release inhibitors result-

224 M.-l. Iskra-Caruana et al. / Journal of Virological Methods 153 (2008) 223–231

Table 1Features of the sets of primers used in molecular tests

Set of primers Product size (bp) Specificity

Oligo 1n: 5′-ATG GTH TGG TGY ATH GAR AAY GG-3′; Oligo 2n: 5′-TGC TGC KGC YTT CAT YTG-3′ 327 Potyvirus (Marie-Jeanneet al., 2000)

B ′ ′ ′ TC GA ′

B AGC C

B GC CC

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17pmw2

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ract 1: 5 -GAC ATC ACC AAA TTT GAA TGG CAC ATGG-3 ; Bract 2: 5 -CCA TTA TCA Cract N1: 5′-GGR ACA TCA CCA AAT TTR AAT GG′-3′; Bract NR: 5′-GTG TGC YTC TCT

ract N2: 5′-ACA TGG AGT ATG ATG GAT AAGG-3′; Bract NR: 5′-GTG TGC YTC TCT A

f inhibitory plant compounds and could also improve the detec-ion of low titers of virus especially in banana plants (Harper etl., 1999; Mumford and Seal, 1997; Wetzel et al., 1992). A mul-iplex immunocapture-RT-PCR has also been developed to detectimultaneously the main three viruses that infect banana: BBTV,ucumber mosaic virus (CMV) and BBrMV (Sharman et al., 2000).nfortunately, although this assay is perfectly suited for large-scaleetection, it is difficult to set up and to reproduce during routineurveys.

This paper describes the development of an immunocapturene-step RT-PCR assay that is sensitive, specific for the detectionf a broad range of BBrMV isolates and suitable to quarantine andermplasm detections where the rate of virus in samples tested aresually very low. In this publication, we describe a non-describedtrain of BBrMV and evaluate the potential of available BBrMV toolso detect it. Two new sets of primers were designed and are usedn RT-PCR and IC-RT-PCR assays with two commercial One-step RT-CR kits. Finally, the applicability of these new tests is suggested asotential international standards for indexing BBrMV.

. Materials and methods

.1. Plant material

All leaf samples were harvested from banana accessionsaintained under tropical greenhouse conditions at CIRAD inontpellier, France. Bioversity International provided in 1994 five

eld infected banana plants showing typical BBrMV symptomsrom India (cv. Bluggoe (ABB) and cv. Pisang Mas (AA)) and fromhilippines (cv. Teeb Khom (ABB), cv. Cardaba (AAB) and cv. TalanAAA)). These field isolates including AGDIA positive control haveeen characterized as co-infected by BanMMV and BBrMV asescribed in Caruana and Galzi (1998). A single BBrMV infectionas obtained by aphid transmission from the Philippine isolate

cv. Cardaba) to virus-free banana, cv. Cavendish subgroup (AAA),nder laboratory conditions at CIRAD in Montpellier. Symptoms inhe leaves developed 3 weeks after aphid transmission. The infec-ion occurred rapidly in banana plants until they expressed severalypical streaks on the flower bracts 12 months later. The infectedanana plants were characterized by electron microscopy, molec-lar and serology analyses as singly infected by a BBrMV straineferred to as BBrMV ‘Ref’ (Iskra-Caruana, unpublished results). Allanana plants were vegetatively propagated and BBrMV infectionas checked both by observing plants for typical BBrMV symptoms

nd using the BBrMV ELISA test. For further molecular analy-es, the Indian BBrMV isolates used were the infected banana cv.isang Mas.

.2. Preparation of fresh and lyophilized leaf extracts and totalNA extracts

The last emerging leaf sample is tested for the presence ofBrMV using ELISA, RT-PCR and IC-RT-PCR techniques. Plantsxtracts were prepared by grinding 0.5 g of fresh banana leaf tis-ue in 5 ml of grinding buffer (PBS–tween buffer: 136 mM NaCl,

(fifrl

T CAA TAC CTC ACAG-3 604 BBrMV (Rodoni et al., 1999)CT GTT-3′ 280 BBrMV (Bract N1 is a slight

modification of Bract 1)T GTT-3′ 260 BBrMV

.4 mM KH2PO4, 2.6 mM KCl, 8 mM NA2HPO4, 0.05% Tween-20, pH

.4, supplemented with 2% polyvinylpyrrolidone, 0.2% sodium sul-hite, 0.2% BSA) using a manual bead grinder and plastic bags withesh (SEDIAG, 21078 Dijon Cedex, France). The ground materialas transferred into microtubes and centrifuged at full speed formin. The supernatant was collected and used for testing.

0.2 g of fresh banana leaf tissue was lyophilized using ChristLPHA 1-4 machine (Bioblock Scientific, France). Plants extractsere prepared by grinding 0.1 g of lyophilized banana leaf tissue

n 5 ml of grinding buffer as described above.Total RNA was extracted from 0.1 g of fresh banana leaf samples

sing the RNeasy plant minikit (QIAGEN©, Courtaboeuf, France)ccording to the manufacturer’s instructions.

.3. Diagnostic tests

.3.1. ELISAA commercial ELISA kit (AGDIA, USA), specific for BBrMV, was

sed. It is a DAS-ELISA protocol with polyclonal antibodies for bothoating antibodies and conjugate antibodies. The test was per-ormed according to the manufacturer’s instructions and colourevelopment was detected at 405 nm using an ELISA plate readerDynatech MRX).

.3.2. One-step-RT-PCRThe primers used for RT-PCR and their specificities are

isted in Table 1. Bract N1, Bract N2 and Bract NR primersere designed on the most conserved part of the CP gene

egion after sequence alignment of the following 23 isolates ofBrMV: GenBank accessions nos. AF071582, AF071584, AF071585,F071586, AF071587, AF071588, AF071589, AF071590, AY494979,Y529121, AY776327, AY953427, BBU88882, BBU88883, BBU88884,BU88885, BBU88886, BBU88887, EF654655, EU009210, EU414267,77912, 1091140.

Two efficient One-step RT-PCR kits were used: QIAGEN OneStepT-PCR system (QIAGEN©—Ref 10210) and Titan One Tube RT-PCRystem (Roche©—Ref 11 855 476 001). All tests were performedccording to the manufacturers’ instructions using 1 �l of RNA forhe first-strand cDNA synthesis. For the QIAGEN OneStep process,he PCR reaction mix (25 �l) contained 1 �l of each primer (10 �M)sed, 0.5 �l of dNTP Mix (2.5 mM), 5 �l of QIAGEN OneStep RT-PCRuffer 5×, 1 �l of QIAGEN OneStep RT-PCR enzyme mix and 0.25 �lf RNase inhibitor. The first-strand cDNA was synthesized by incu-ating at 50 ◦C for 30 min followed by an initial PCR denaturationtep of 15 min at 94 ◦C and 30 PCR amplification cycles of 1 min at4 ◦C, 1 min at 57 ◦C, 30 s at 72 ◦C, and a final extension of 5 min at2 ◦C.

For the Titan One Tube process, the PCR reaction mix (25 �l) con-ained 1 �l of each primer (10 �M) used, 1 �l of dNTP Mix (2.5 mM),�l of Titan One Tube RT-PCR Buffer 5×, 1.25 �l of DTT solution

100 mM), 0.5 �l of enzyme mix and 0.25 �l of RNase inhibitor. Therst-strand cDNA was synthesized by incubating at 50 ◦C for 30 min

ollowed by an initial PCR denaturation step of 15 min at 94 ◦C, a firstun of 10 PCR cycles of 15 s at 94 ◦C, 30 s at 57 ◦C, 30 s at 68 ◦C, fol-owed by a second run of 30 PCR cycles of 15 s at 94 ◦C, 30 s plus 5 s

M.-l. Iskra-Caruana et al. / Journal of Virological Methods 153 (2008) 223–231 225

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ig. 1. Symptoms of the BBrMV ‘Ref’ strain in cv. Cavendish. (A) Light white mosaicndicate leaves of rows 1, 3 and 8 after the last emerging leaf (row 0). (C) Typical darbserved under the electron microscope. (For interpretation of the references to col

or each PCR cycle all at 57 ◦C, 30 s at 60 ◦C, and a final extension of0 min at 68 ◦C.

Amplicons were visualized after migration of 10 �l of PCR prod-cts (3 �l for Titan reaction) on a 1–1.5% agarose gel in 0.5× TBE45 mM Tris–borate, 1 mM EDTA, pH 8). The gel was stained withthidium bromide and amplified bands were visualized under UVight.

.3.3. IC One-step-RT-PCRPrior to RT-PCR amplification, an immunocapture of particles

as performed using BBrMV polyclonal antibodies from AGDIA.amples were treated as described for ELISA up to, and including,he three washes with PBS–tween following incubation of the sapxtract for 3 h at room temperature or overnight at 4 ◦C using ster-le RNAse-free polypropylene thin-walled microfuge tubes. Tubes

ere washed four to five times with sterile PBS–tween followed bywo washes with DEPC-treated sterile water. One-step RT-PCR waserformed in the same tubes as described above.

. Results

.1. BBrMV symptoms observed on cv. Cavendish (AAA)

Field symptoms of BBrMV are usually described as a yellowosaic of the leaves and of the pseudo-stem, which turns red,ith the typical final symptom of dark red streaks on the bracts

f the banana inflorescence. All BBrMV field isolates maintainednder greenhouse conditions at CIRAD in Montpellier showed allhese symptoms. All were co-infected with BanMMV detected by

e lamina of the last emerging leaf. (B) Symptomless infected banana plant. Arrowstreaks in the bracts of the inflorescence (arrows). (D) Purified BBrMV ‘Ref’ particlesthis figure legend, the reader is referred to the web version of the article.)

osaic sometimes appeared in the limb of the newly emerging leafFig. 1A), before completely disappearing 24–48 h later. Infectedanana plants remained symptomless (Fig. 1B) until the develop-ent of the banana inflorescence with typical mosaic symptoms

n the bracts (Fig. 1C) and on the fruits. Purified viral particlesppeared filamentous and flexuous under the electron microscopeFig. 1D) and were similar in size and shape to potyvirus particles.

.2. Evaluation of existing tests for the detection of BBrMV

Available serological and molecular tools to detect BBrMV wereompared in terms of specificity and sensitivity. AGDIA©sells anLISA kit using a BBrMV polyclonal antibodies (Thomas et al.,997). Rodoni et al. (1999) developed PCR-based molecular testssing specific Bract 1/2 primers: a RT-PCR and an IC-RT-PCR. Theotyvirus degenerate primers Oligo 1n/2n developed by Marie-

eanne et al. (2000) were also tested in this comparative study toonfirm the detection of BBrMV which is a potyvirus. ELISA, RT-PCRnd IC-RT-PCR tests were undertaken on several plant samples withr without BBrMV infection (described in Section 2). The resultsre summarized in Table 2. They show ELISA and PCR tests usingotyvirus degenerate primers Oligo 1n/2n enabled detection of allBrMV-infected samples, but the specific BBrMV primers (Bract/2) did not react against the Indian BBrMV isolates and BBrMVRef” plants.

Next, to evaluate their efficiency, using Oligo 1n/2n, ELISA andT-PCR were performed on the same set of samples selected accord-

ng to their differing viral titers as indicated by the OD recorded inLISA (Fig. 2B). Fresh and frozen Indian isolates showed a compara-

ow titer, was not detected by ELISA (data not shown). The compar-son showed a similar level of detection related to the viral titers,

Table 2Comparison of available tools for BBrMV detection

Tests ELISA RT-PCR IC-RT-PCR

AGDIA kit Bract 1/2a Oligo 1n/2nb Bract 1/2 Oligo 1n/2n

Philippine BBrMV isolate 509� + + + + +AGDIA banana positive control + + + + +Indian BBrMV isolate + − + − +BBrMV ‘Ref’ strain + − + − +Healthy cv. Cavendish plant − − − − −WMV2 infecting vanilla − − + − +SCSMV-infected sugarcane − − − − −PVY-infected tomato − − + − +

− ugarcJ

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: Negative result; +: positive result; WMV2: Watermelon mosaic virus 2; SCSMV: s.E. Thomas.

a Primers designed for specific detection of BBrMV (Rodoni et al., 1997).b Primers designed to detect potyviruses (Marie-Jeanne et al., 2000).

ncluding no detection of both BBrMV ‘Ref’ strain plant number IViluted to 1 × 10−2 (ELISA and RT-PCR) and plant number III (RT-CR) (Fig. 2A). It was concluded therefore that the ELISA is as reliables RT-PCR using Oligo 1n/2n to detect BBrMV but does not detectow levels of BBrMV infection in banana plants.

.3. Design of new sets of primers to detect BBrMV

Alternative specific primers were designed for broad spectrumetection of BBrMV in PCR-based molecular test since Bract 1/2

ig. 2. Comparison between RT-PCR and ELISA tests to detect BBrMV in banana sam-les. (A) RT-PCR using potyvirus degenerate primers Oligo 1n/2n. (B) ELISA graph ofD recorded at A405 nm 1–4 h after enzyme substrate was added. The same samplesere used in both tests—1f: fresh sample of Indian BBrMV isolate; 1c: frozen sam-le of Indian BBrMV isolate; 2: BBrMV ‘Ref’ plant number IV; 3: BBrMV ‘Ref’ plantumber IV diluted to 1 × 10−2; 4: virus-free cv. Pisang Klutuk Wulung; 5: BBrMV

Ref’ plant number III; 6: H2O; M: 1 kb ladder (Invitrogen©).

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ane streak mosaic virus; PVY: potato virus Y; �: BBrMV isolate kindly given by Dr.

rimers did not detect all BBrMV isolates and potyvirus degener-te primers Oligo 1n/2n Oligo 1n/2n were not sensitive enough toetect low levels of viral infection and were not specific to BBrMV.

Sequence alignment of the entire CP-coding region was carriedut between the 23 isolates of BBrMV available in GenBank includ-ng the sequence of the BBrMV 509 isolate BBrMV India isolateGenBank accession no. 1091140) and BBrMV ‘Ref’ strains (Gen-ank accession no. EU414267) in order to achieve broad detection ofBrMV. Three primers are designed within the fragment amplifiedy the potyvirus degenerate primers Oligo 1n/2n and based on theonsensus region. Two primers (Bract N1 and Bract N2) located inhe 5′ region and one primer (Bract NR) located in the 3′ region wereelected. Primer Bract N1 was degenerate at two sites, Bract NR atne, and Bract N2 at none. The expected amplification productsere of 260 and 280 bp with the two sets of Bract N1/NR and Bract2/NR primers, respectively. Several PCR conditions were testedith the Qiagen©OneStep kit including a temperature test ranging

rom 55 to 60 ◦C using banana leaf extracts infected by the IndianBrMV isolate used above (Fig. 2, sample 1) and diluted severalime after grinding to 1 × 10−8. We recorded a specific and sensi-ive detection for both sets of primers from 57 and 58 ◦C as shownn Fig. 3, followed by a significant extinction at 60 ◦C. The inten-ity of the amplified band obtained with Bract N2 was greater thanhat obtained with Bract N1 for the same samples. Their specificityas confirmed by RT-PCR against either several viruses infectinganana plants such as BSV, BanMMV, BBTV and CMV, or otherotyviruses infecting plants such as those infecting vanilla (Water-elon mosaic virus 2—WMV2) and potatoes (Potatoes virus Y—PVY)

data not shown). An annealing temperature of 57 ◦C was selectedor use with both primer combinations.

.4. Comparison of the new and existing primers for the detectionf BBrMV

The first comparison concerned RT-PCR using the Qiagen©On-Step kit. Primer sets Bract N1/R and Oligo 1n/2n were comparedsing RNA extracted from banana samples used previously for theLISA above (Fig. 2). They were representative of different levels ofirus infection ranging from high (sample 1) to not detectable byLISA (sample 3). Each RNA extract was diluted to 1 × 10−1, 1 × 10−2

nd 1 × 10−4 (Fig. 4A–C, respectively).The reaction with Bract N1/NR primers (Fig. 4, panel 2) is

tronger than the reaction with Oligo 1n/2n primers (Fig. 4, panel

F V setN ilutedc rogen©

ig. 3. Comparison of IC-RT-PCR temperatures using the two newly designed BBrM1/NR primers; (2) Bract N2/NR primers; 1–4: leaf samples of Indian BBrMV isolate dv. Pisang Klutuk Wulung; 6: virus-free cv. Cavendish; 7: H2O; M: 1 kb ladder (Invit

A decreasing pattern of reaction was recorded with both primers

ig. 4. Comparison between the degenerate potyvirus and the Bract Nat1/NR primers in Ramples 1f: fresh sample of Indian BBrMV isolate; 1c: frozen sample of Indian BBrMV is× 10−2; 4: virus-free cv. Pisang Klutuk Wulung; 5: BBrMV ‘Ref’ plant number III; 6: H2O;xtracts, respectively. (1, 2) RT-PCR using degenerate Oligo 1n/2n and Bract N1/NR, respec

s of primers and the QIAGEN©OneStep kit. (A) 57 ◦C; (B) 58 ◦C; (C) 60 ◦C. (1) Bractafter grinding to 1 × 10−5, 1 × 10−6, 1 × 10−7 and 1 × 10−8, respectively; 5: virus-free).

sample 5) (Fig. 4, panel 2A) while Oligo 1n/2n primers showed no

The second comparison concerned IC-RT-PCR using the QIA-EN©OneStep kit. Immunocapture was performed on the banana

eaf extracts used previously for the ELISA above (Fig. 2). Surpris-

T-PCR using the QIAGEN©OneStep kit. Total RNA was extracted from banana plantolate; 2: BBrMV ‘Ref’ plant number IV; 3: BBrMV ‘Ref’ plant number IV diluted toM: 1 kb ladder (Invitrogen©). A–C: 1 × 10−1, 1 × 10−2 and 1 × 10−4 dilutions of RNAtively.

Fig. 5. Comparison between degenerate potyvirus and Bract N1/NRprimers in IC-RT-PCR using the QIAGEN©OneStep kit. Ground banana leaf 1f: fresh sample of IndianBBrMV isolate; 1c: frozen sample of Indian BBrMV isolate; 2: BBrMV ‘Ref’ plant number IV; 3: BBrMV ‘Ref’ plant number IV diluted to 1 × 10−2; 4: virus-free cv. Pisang KlutukW 6 dil1 e Olig1

ulung; 5: BBrMV ‘Ref’ plant number III; 6: RNA of BBrMV ‘Ref’ sample; 6′: sample× 10−2 dilutions of grinding samples, respectively. (1, 2) RT-PCR using degenerat–4 h after enzyme substrate was added.

ngly, we recorded a similar reaction in the two sets of primers evenhough the reaction was stronger for Bract N1/NR primers than forligo 1n/2n primers (Fig. 5). Unfortunately, neither detected the

ow and undetectable rates of viral infection that were detected byT-PCR and Bract N1/NR primers (Fig. 5, samples 3 and 5).

To increase the efficiency of IC-RT-PCR detection, the Titan One

uted to 1 × 10−2; 7: H2O; M: 1 kb ladder (Invitrogen©). A–C: 1 × 10−1, 2 × 10−1 ando 1n/2n and Bract N1/NR, respectively. (3) ELISA graphs of OD recorded at A405 nm

.5. Application of IC One-step RT-PCR for the routine detection ofBrMV

.5.1. Ability to detect BBrMV in banana plant in routine testsThe ability of the IC One-step RT-PCR was tested in order to

efine the most appropriate part of the plant to use in routine tests

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Table 3Feasibility of using the IC One-step RT-PCR technique for the routine detection ofBBrMV

Samples ELISA IC-RT-PCR

N1/NR N2/NR

CIRAD CIRAD LPV 972

Titan Qiagen Titan Qiagen Qiagen

1: cv. SA − − − − − −2: cv. PKW − − − − − −3: cv. Cavendish − − − − − −4: BBrMV ‘Ref’ number I − +/− + + + +5: BBrMV ‘Ref’ number III + ++ ++ +++ ++ +6: BBrMV ‘Ref’ number V ++ +++ ++ +++ ++ +7: Indian BBrMV isolate +++ ++ + + + +8: cv. PKW − − − − − −9: cv. Cavendish − − − − − −A: 1 + 2 + 3 + 4 + 5 + ++ ++ +++ ++ ++B: 7 + 8 + 9 + 3 + 2 +++ ++ + +++ ++ ++C: 2 + 6 + 3 + 9 + 8 + +++ ++ +++ ++ +D: 3 + 9 + 2 + 8 + 4 − + + ++ + +E: 3 + 9 + 2 + 8 + 1 − − − − − −A + E + +++ ++ +++ ++ ++B + E +++ + + + + +C + E + +++ ++ +++ ++ ++D + E − − + − +/− +A + D − +++ +++ ++ + +B + C ++ ++ +++ ++ ++ ++E + E − − − − − −

ig. 6. IC-RT-PCR using Titan One Tube kit and the two Bract N1/NR and N2/NRrimers. (1–6) BBrMV ‘Ref’ plant number IV diluted to 1 × 10−5, 1 × 10−7, 1 × 10−8,× 10−9 and 1 × 10−10; 6: virus-free cv. Pisang Klutuk Wulung; 7: virus-free cv.avendish; 8: H2O; M: 1 kb ladder (Invitrogen©). (A and B) IC-RT-PCR using theract N1/NR primers and the Bract N2/NR primers, respectively.

etection succeeded whatever part of the plant tested and wasimilar in both fresh and lyophilized packaging (Fig. 7A). BBrMVnfection of the three symptomless leaves selected according toheir row was well detected in the same symptomless plant (Fig. 7B,amples 1–3) and the sample from youngest leaf was detected even

.5.2. Ability to detect BBrMV during routine diagnosisThe different assays used to detect BBrMV were evaluated in

omparison with each other and with ELISA to establish the most

ig. 7. BBrMV detection on several parts of banana plant by IC-RT-PCR using Titanne Tube kit and the Bract N2/NR primers. (A) 1: banana bract of BBrMV ‘Ref’ plantumber III; 2: lyophilized BBrMV ‘Ref’ plant number III; 3: lyophilized sample 1;: H2O; 5: virus-free cv. Pisang Klutuk Wulung; 6: lyophilized virus-free bananaract; 7: lyophilized Indian BBrMV isolate; 8: RNA of Indian BBrMV isolate; M: 1 kb

adder (Invitrogen©). (B) 1–3: leaves 1–3, respectively, of BBrMV ‘Ref’ plant numberV; 4–6: samples 1–3 diluted to 1 × 10−6; 7–9: samples 1–3 diluted to 1 × 10−10; 10:

T + AGDIA ++ ++

aMBeabtIwpTssrwAtdiTebst

LISA: +, low infection rate (OD: 0.150); ++, medium infection rate (0.2 > OD > 0.4);++, high infection rate (OD > 1.5). IC-RT-PCR: the number of crosses is proportionalo the size and the intensity of the amplified PCR fragment. −: No detection.

ppropriate assay to use for routine BBrMV diagnosis in either theusa germplasm or banana plants from the field. Five individual

BrMV-infected banana samples with undetectable up to high lev-ls of infection, and three virus-free banana leaves were selectednd lyophilized for the blind test. Final samples were composed ofanana samples alone and of bulked samples after grinding, con-aining 5 and 10 banana samples as described in Table 3. ELISA andC-RT-PCR using both QIAGEN©OneStep and Titan One Tube kitsere performed in two separate laboratories one at CIRAD in Mont-ellier and one (LPV 972) in Martinique. Results are summarized inable 3. The two techniques reliably detected all BBrMV-infectedamples even when they were bulked with nine other virus-freeamples. However, even though the results of the ELISA were cor-ect, samples 4, D and A + D were not detected at all (Table 3),hereas they were found to be infected by IC One-step RT-PCR.similar detection by IC One-step RT-PCR was observed whatever

he kit we used, except for the (D + E) mixed sample, which was onlyetected by the QIAGEN©OneStep kit. The differences were mainly

n band intensity: stronger amplification was observed with theitan One Tube kit (Fig. 8). Bract N2/NR primers seemed to be morefficient than Bract N1/NR primers with the two kits given a strongand. IC One-step RT-PCR using Bract N2/NR primers appeared to beturdy since the LPV 972 laboratory in Martinique obtained exactlyhe same results as the lab in Montpellier.

. Discussion

All symptoms of BBrMV are reported as occurring permanently

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ig. 8. Comparison of the Titan One Tube kit and the QIAGEN©OneStep kit by IC-RB) QIAGEN OneStep kit. 1: Virus-free cv. Sa; 2 and 8: cv. virus-free Pisang Klutuk W: number III; 6: number V; 7: Indian BBrMV isolate; 10: H2O; 11: AGDIA banana po

as described for the first time, emphasizing the importance ofhe efficiency of detection tests to diagnose infection as early asossible. In fact, the strain called BBrMV ‘Ref’ was selected byphid transmission from a field Philippine BBrMV-infected bananalant containing both BBrMV and BanMMV. The BBrMV ‘Ref’ strain

nduces an infection within the plant that fluctuates with the sea-on, and the infected plant always appears to be symptomlessxcept for the last emerging leaf and at the final growing step whenypical symptoms appear on all the bracts produced. We assumehat the BBrMV symptoms described up to now referred to a mixednfection with BanMMV (Iskra-Caruana, unpublished results) orccurred in cooking banana only.

Using ELISA, the current international standard for indexingBrMV, cyclic variations were observed in the virus titers of theBrMV ‘Ref’ in our banana plants over several years. To overcomehis problem, it was decided to extend the reading time of ELISAest from 1 to 4 h to improve the detection thresholds. Unfortu-ately, our results showed that even though the polyclonal serumeveloped by Thomas et al. (1997) is efficient and consequentlyetection by ELISA is generally satisfactory, it could not detect the

ower viral concentrations existing at the early stages of infectionn symptomless but infected plants, in Musa germplasm as well asn vitro banana plantlets. In this study we demonstrated that RT-CR using the Bract 1/2 primers lack broad spectrum detection ofBrMV since they did not detect both the Indian BBrMV isolate andhe BBrMV ‘Ref’ strain as also observed by Sharman et al. (2000),nd that RT-PCR using the Oligo 1n/2n primers is as sensitive as

Finally, an efficient IC One-step RT-PCR specific to the diagno-is of BBrMV was developed with newly designed sets of primers.hese primers have only been tested against a few BBrMV isolates

using the Bract N2/NR primers in BBrMV blind diagnostic. (A) Titan One Tube kit;; 3 and 9: virus-free cv. Cavendish BBrMV ‘Ref’ infected banana plant; 4: number I;control; M: 1 kb ladder (Invitrogen©).

uch as Indian, Philippine and AGDIA BBrMV-infected isolates, buthey are designed on a conserved part of the viral genome after anlignment of more than 23 different sequences available in Gen-ank.

By comparing RT-PCR and immunocapture-RT-PCR, the advan-age in BBrMV detection of introducing an immunocapture stepefore the RT-PCR process was established, even if RT-PCR seemso be little bit more sensitive than the other tests. Indeed, this step

eans RNA extraction can be avoided, which often damages RNAiruses especially those present at low concentrations, and com-ined with the feature of banana plants themselves containing a lotf tannins and latex. In this case, the final viral particles in tubes arenriched by captured polyclonal antibodies whilst PCR inhibitorsre eliminated from the samples during the immunocapture step,ll of which gives a significant advantage to IC-RT-PCR in revealingow concentrations of viruses in plants.

This assay (using either the Bract N1/NR or the Bract N2/NRrimers) is extremely sensitive in plant detection since it allowspecific detection of BBrMV in plant extracts diluted to 1 × 10−10

n some case versus only 1 × 10−5 reported by Rodoni et al. (1999)sing the Bract 1/2 primers.

Two commercial kits were compared to develop the molecularest, the QIAGEN©OneStep kit and the Titan One Tube kit to increaseignificantly the PCR signal. In comparison the Titan One Tube kitame out best in terms of band intensity and threshold of detectionFig. 8). The comparison in routine diagnoses by blind detection ofBrMV in several samples constituted by bulking 5 and 10 plants

he Titan One Tube kit, including better detection of very low viraloncentrations (Fig. 8, sample (D + E)). These are interesting find-ngs for BBrMV indexing because of the high cost of the Titan One

Virolo

Teiosec

M.-l. Iskra-Caruana et al. / Journal of

ube kit and because of the possibility to bulk samples when thexpected rate of positive samples is low. Finally, the last emerg-ng leaf remains the most suitable part of plant for early detectionf infection and that lyophilized packaging is the suitable way oftoring the banana samples. The fact this test can be transferredasily and set up by another team in tropical countries is also aonsiderable advantage.

This IC One-step RT-PCR assay using the Bract N2/NR primersnd the QIAGEN OneStep kit could be promoted as the new refer-nce test for routine large-scale BBrMV indexation such as surveys,ndexing Musa germplasm as well as for in vitro banana plantletsntroduced in virus-free countries.

cknowledgements

This work was supported by the Ministère Francais de’Agriculture et de la Pêche, Direction Générale de l’Alimentation,ous Direction de la Qualité et de la Protection des Végétaux. Wehank the LPV 972 laboratory for their collaboration and particularlyatricia Rouet and Jean Iotti.

eferences

aruana, M.L., Galzi, S., 1998. Identification of uncharacterized filamentous viralparticles on banana plants. Acta Horticulturae 490, 323–335.

ambley, C.F., Thomas, J.E., 2001. Molecular characterisation of Banana mild mosaicvirus, a new filamentous virus in Musa spp. Archives of Virology 146, 1369–1379.

arper, G., Dahal, G., Thottappilly, G., Hull, R., 1999. Detection of episomal bananastreak badnavirus by IC-PCR. Journal of Virological Methods 79, 1–8.

enyon, L., Magnaye, L.V., Warburton, H., Herradura, L., Chancellor, T., Escobido, E.,Foot, C., 1996. Epidemiology and control of banana virus diseases in the Philip-pines. Final technical report Project A0217/X0258. In: N.d.f.i.d.o.c.p.p. Project(Ed.), Chatham Maritime, UK.

gical Methods 153 (2008) 223–231 231

agnaye, L.V., Espino, R.R.C., 1990. Note: banana bract mosaic, a new disease ofbanana. I. Symptomatology. The Philippines Agriculturist 73, 55–59.

arie-Jeanne, V., Ioos, R., Peyre, J., Alliot, B., Signoret, P., 2000. Differentiation ofpoaceae potyviruses by reverse transcription-polymerase chain reaction andrestriction analysis. Journal of Phytopathology 148, 141–151.

umford, R.A., Seal, S.E., 1997. Rapid single-tube immunocapture RT-PCR for thedetection of two yam potyviruses. Journal of Virological Methods 69, 73–79.

unez, A.R., 1992. Symptomatology, Transmission and Purification of banana bractmosaic virus (BBrMV) in ‘Giant Cavendish’ Banana. University of Philippines, LosBanos.

odoni, B.C., Ahlawat, Y.S., Varma, A., Dale, J.L., Harding, R.M., 1997. Identificationand characterization of banana bract mosaic virus in India. Plant Disease 81,669–672.

odoni, B.C., Dale, J.L., Harding, R.M., 1999. Characterisation and expression of thecoat protein-coding region of banana bract mosaic potyvirus, development ofdiagnostic assays and detection of the virus in banana plants from five countriesin southeast Asia. Archives of Virology 144, 1725–1737.

harman, M., Thomas, J.E., Dietzgen, R.G., 2000. Development of a multipleximmunocapture PCR with colourimetric detection for viruses of banana. Journalof Virological Methods 89, 75–88.

eycheney, P.Y., Acina, I., Lockhart, B.E.L., Candresse, T., 2007. Detection of bananamild mosaic virus and banana virus X by polyvalent degenerate oligonucleotideRT-PCR (PDO-RT-PCR). Journal of Virological Methods 142 (1–2), 41–49.

homas, J.E., Iskra-Caruana, M.-L., 2000. Diseases caused by viruses: bunchy top.In: Jones, D.R. (Ed.), Diseases of Banana, Abaca and Enset. CABI, Oxon, UK, pp.241–253.

homas, J.E., Geering, A.D.W., Gambley, C.F., Kessling, A.F., White, M., 1997. Purifica-tion, properties and diagnosis of Banana bract mosaic potyvirus and its distinctionfrom Abaca mosaic potyvirus. Phytopathology 87, 698–705.

homas, J.E., Magnaye, L.V.M., 1996. Banana Bract Mosaic Disease. Musa Fact SheetNo. 7. INIBAP, Montpellier, France.

homas, J.E., Iskra-Caruana, M.L., Magnaye, L.V., Jones, D.R., 2000a. Diseases causedby viruses: bract mosaic. In: Jones, D.R. (Ed.), Diseases of Banana, Abacà andEnset. CABI, London, pp. 253–263.

homas, J.E., Lockhart, B.E.L., Iskra-Caruana, M.L., 2000b. Diseases caused by viruses:banana mild mosaic. In: Jones, D.R. (Ed.), Diseases of Banana, Abacà and Enset.CABI, London, pp. 275–278.

etzel, T., Candresse, T., Macquaire, G., Ravelonandro, M., Dunez, J., 1992. A highlysensitive immunocapture polymerase chain reaction method for plum poxpotyvirus detection. Journal of Virological Methods 39, 27–37.

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