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Sensors and Actuators B 139 (2009) 429–434

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l homepage: www.e lsev ier .com/ locate /snb

ptimization detection of plant pathogenic bacteria bylectrochemiluminescence polymerase chain reaction method

ie Wei, Baoyan Wu ∗

OE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China

r t i c l e i n f o

rticle history:eceived 4 November 2008eceived in revised form 18 March 2009ccepted 19 March 2009vailable online 31 March 2009

eywords:

a b s t r a c t

Plant pathogenic bacteria spread all over the world, causing a great deal of economic loss. This study hasdeveloped a novel method for rapid detection of plant pathogenic bacteria by electrochemiluminescencepolymerase chain reaction (ECL-PCR) using two universal probes, a biotin-probe and a Ru(bpy)3

2+ (TBR)-probe. Biotin-probe sequence is complementary to the universal sequence of anti-sense primer, and TBR-probe sequence is the same as the universal sequence of sense primer. So the amplified PCR products canhybridize with TBR-probe and biotin-probe. After hybridizing, PCR products are captured by streptavidin

lectrochemiluminescenceolymerase chain reactionniversal probelant pathogenic bacteria

coated magnetic bead through the biotin–streptavidin linkage, and then TBR reacts with tripropylamine toemit light for detection of plant pathogenic bacteria. This proposed method is applied to detect Fusariumoxysporum f. sp. Cubense and Xanthomonas oryzae pv. Oryzae. The results show that the method cansuccessfully identify the plant pathogenic bacteria in the infected samples and these results are consistentwith the results of gel electrophoresis. Importantly, this study can be used as an illustration for detectingvarious plant pathogenic bacteria and provides a feasible approach on developing ECL sensors to meetthe demand of rapid detection of pathogenic bacteria, fungi, and viruses.

. Introduction

Plant pathogenic bacteria cause many serious diseases of plantshroughout the world. Severe reduction of growth and yield oflants and vectors infected by plant pathogenic bacteria signifi-antly affects economy. Among all the plant pathogenic bacteria,usarium oxysporum f. sp. Cubense and Xanthomonas oryzae pv.ryzae are the two most representative ones. Fusarium wilt causedy F. oxysporum f. sp. Cubense is one of the most destructive diseasesf banana worldwide [1]. The typical symptoms include wilting andeath of the leaves, followed by death of the whole plant. X. oryzaev. oryzae, the primary cause of bacterial blight of rice, is one ofhe most important pathogens of rice in most of the rice-growingountries [2]. Bacterial blight of rice is destructive to high-yieldingultivars in both temperate and tropical regions especially in Asia.herefore, it is significant to develop an effective method for rapidetection of bacteria and fungus in plants and vectors to ensuregricultural production with security.

Various techniques have been developed for the detection oflant pathogenic bacteria. Microscope, isolated culture, bioassaynd serology technology were widely used in plant pathogenic bac-eria detection, but these methods are not only time-consuming and

∗ Corresponding author. Tel.: +86 20 85217070x8406; fax: +86 20 85216052.E-mail address: wubaoyan@scnu.edu.cn (B. Wu).

925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2009.03.054

© 2009 Elsevier B.V. All rights reserved.

costly, but also unreliable [3]. Enzyme linked immunosorbent assay(ELISA) is the main way to detect pathogenic bacteria, but someextractive in plants can suppress the reaction, which brings a lot ofdifficulties in the detection procedure [4]. A lot of detection assaybased on PCR methodology were developed and applied to identifyand detect the plant pathogenic bacteria [5]. Since Kenten et al. [6]and Blackburn et al. [7] applied DNA probe to ECL, this method hasbeen widely used in DNA analysis [8–11] and various areas rang-ing from chemical analysis to the molecular-level understandingof biological processes [12–15]. ECL combines chemiluminescenceand electrochemistry, which is a chemiluminescent reaction ofspecies generated electrochemically at an electrode surface. Ini-tially, TBR and TPA are oxidized at the surface of an anode toproduce Ru(bpy)3

3+ and TPA+*. TPA+* immediately loses a protonand becomes a powerful reducer (TPA*). When TPA* and Ru(bpy)3

3+

react, the latter enters an excited state (Ru(bpy)32+*) by a high

energy electron transfer from TPA*. Relaxation of Ru(bpy)32+* to the

ground state (TBR) results in a light emission at 620 nm [16]. Notice-ably, TBR is not consumed during the reaction and may be oxidizedand excited repeatedly, if there is excessive TPA in the buffer.

Recently, we have developed a highly sensitivity ECL-PCR tech-

nique described in our previous articles for nucleic acid detection[17–20]. The key idea of the paper is to develop a novel method forrapid detection of plant pathogenic bacteria by introducing a pairof universal probes. Ribosomal DNA (rDNA) is the most conservedregion in the genome, with capabilities of phylogenetic divergence

430 J. Wei, B. Wu / Sensors and Actuators B 139 (2009) 429–434

Table 1Primers and probes used in this study.

Name Sequence (5′–3′)

Fusarium oxysporum f. sp. Cubense Sense primer 5′-TAACTGAATAGACTAAGACAGCAAAATGCGATAAGTAAT-3′

Anti-sense primer 5′-CTAATCAACGACCTTGTATCTATTCCTACCTGATCCGAGG-3′

Xanthomonas oryzae pv. Oryzae Sense primer 5′-TAACTGAATAGACTAAGACGCATGACGTCATCGTCCTGT-3′

U

[tcysspXbml

2

2

pncAwse

R(pX

Anti-sense primer

niversal probes TBR-probeBiotin-probe

21]. rDNA as a target has been widely used in bacteria detec-ion and identification, which has become a most effective andommonly used molecular marker of bacteria phylogenetic anal-sis [22–24]. So we amplify the internal transcribed spacer (ITS)equence in rDNA of F. oxysporum f. sp. Cubense and 16S–23S rDNApacer regions of X. oryzae pv. Oryzae for ECL-PCR-mediated plantathogenic bacteria detection. With F. oxysporum f. sp. Cubense and. oryzae pv. oryzae as the model plant pathogenic bacteria, andiotin-probes and TBR-probes as the universal probes, this novelethod to rapid detection of plant pathogenic bacteria is estab-

ished.

. Materials and methods

.1. Materials

Taq DNA polymerase, dNTP and 600 bp DNA Ladder wereurchased from Shanghai Sangong Biological Engineering & Tech-ology Services Co. Ltd. (SSBE), China. �-Mercaptoethanol andetyltrimethyl ammonium bromide (CTAB) were products ofMRESCO, Netherlands. The streptavidin coated magnetic beadsere acquired from MACS, Germany. TPA and the chemicals to

ynthesize the Ru(bpy)32+ N-hydroxysuccinimide ester (TBR-NHS

ster) were from Sigma–Aldrich Chemical Company, USA.

F. oxysporum f. sp. Cubense was obtained from College of Nature

esources and Environment, South China Agricultural UniversityGuangzhou, China). Both infected and uninfected banana leaf sam-les were collected from banana plantation in Panyu, Guangzhou.. oryzae pv. Oryzae and infected rice leaf samples were obtained

Fig. 1. The basic principle of this ECL-PCR method

5′-CTAATCAACGACCTTGTATCCTCGGAGCTATATGCCGTGC-3′

5′-TBR-TAACTGAATAGACTAAGAC-35′-biotin-GATACAAGGTCGTTGATTAG-3′

from institute of Plant Protection, Academy of Agricultural Sciences(Guangzhou, China).

Primers of F. oxysporum f. sp. Cubense and probes were designedby our lab, and primers of X. oryzae pv. Oryzae has been reportedbefore [5]. The rDNA sequence of F. oxysporum f. sp. Cubense hasbeen assigned with a GenBank accession no. EU780660, availableat http://www.ncbi.nlm.nih.gov. On the basis of these nucleotidesequences, sense primer and anti-sense primer, were designed forspecific amplification of ITS sequence in rDNA of F. oxysporum f.sp. Cubense using the Primer Premier 5.0 software. All primers andprobes were synthesized by SSBE (Table 1). Universal sequences inprimers have been underlined, which indicate the region of sensorprimers is the same as the TBR-probe sequences, and anti-senseprimers are complementary to biotin-probe sequences. The probeswere labeled with biotin by SSBE and TBR by our lab, respectively.

2.2. Principle

Fig. 1 shows the basic principle of ECL-PCR for plant pathogenicbacteria detection, which involves the following four steps:extraction of genome DNA from plant pathogenic bacteria, PCRamplification of the targeted sequence, hybridization with the TBR-probe and biotin-probe, and ECL detection of targeted sequence.First, genome DNA was extracted from F. oxysporum f. sp. Cubense,

X. oryzae pv. oryzae, infected samples and healthy samples usingCTAB method, respectively. Second, the targeted oligonucleotidesequence, the ITS sequence of F. oxysporum f. sp. Cubense and16S–23S rDNA spacer regions of X. oryzae pv. Oryzae were amplifiedby PCR. Third, the amplified PCR products were hybridized with the

for detection of plant pathogenic bacteria.

Actuators B 139 (2009) 429–434 431

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role in the reaction between TBR and TPA. When the temperaturefluctuates around the room temperature, it has such a small impacton the reaction so that it can be ignored [17]. So, all the experimentsin this paper were progressed under the room temperature.

J. Wei, B. Wu / Sensors and

BR-probe and biotin-probe. Fourth, through the biotin-probe, theCR products were caught by streptavidin coated magnetic beadsased on the highly selective biotin–streptavidin linkage [7,25].hen ECL detection of PCR products was executed through theBR-probe, which would emit photons on the anode surface. Thehotons are captured by an ECL detection system and can be readut through Labview software, which reflects the quantity of theybridized PCR products [26].

.3. DNA extraction and PCR amplification

CTAB method for sample extraction and purification reported byipp et al. was used in this study [27]. All the samples were grindedith sterile mortar and extracted with CTAB precipitated, treatedith chloroform, and precipitated with isopropanol to obtain aurified DNA matrix.

The PCR reaction of F. oxysporum f. sp. Cubense and X. oryzae pv.ryzae was carried out in 25 �L mixtures containing 1 �L of sam-le DNA, 2.5 �L 10× Taq polymerase buffer, 0.5 �L dNTP (10 mM),5 pmol sense and anti-sense primers, and 2.5 U Taq polymerase.he amplification reaction was performed on thermal circler (PTC-00, MJ Research Inc., USA). Annealing temperature of F. oxysporum. sp. Cubense PCR amplification is 50 ◦C, and the whole reactionontains 35 cycles. Annealing temperature of X. oryzae pv. Oryzae is3 ◦C, and the whole reaction contains 30 cycles.

.4. Hybridization with probes and ECL detection

Hybridizations with biotin-probe and TBR-probe were per-ormed by adding 20 �L of each probe to 10 �L of PCR products.he mixture was incubated for 5 min at 94 ◦C, then 1 h at 50 ◦C for. oxysporum f. sp. Cubense and 1 h at 63 ◦C for X. oryzae pv. Oryzaen the PCR system.

For the sample ECL detection, 20 �L of hybridization productsnd 10 �L of streptavidin coated magnetic beads were added to70 �L TE binding buffer [28,29], and incubated for 30 min at roomemperature with gentle shaking to form the biotin–streptavidininkage. The sample was collected by magnetic separation, follow-ng washed with binding buffer and added into ECL reaction cell.hen, TE buffer containing TPA was also added into the reactionell. A voltage of 1.25 V was applied across the electrodes and theignals of ECL were read out using Labview software. Each sampleas detected six times and analyzed with statistical method.

.5. Apparatus

An ECL detection system has been custom-built [16,17]. Thenstrument is composed of an electrochemical reaction cell, aotentiostat (Sanming Fujian HDV-7C), an ultra high sensitiv-

ty single photon counting module (Channel Photomultiplier,erkinElmer MP-962), a multi-function acquisition card (AdvantechCL-836), a computer and labview software. The electrochemicaleaction cell contains a working electrode (platinum), a counterlectrode (platinum), and a reference electrode (Ag/AgCl).

. Results and discussion

.1. Optimization of ECL detection

Here, the optimal concentration of probe and the pH value of

ybridization buffer were determined. Fig. 2 shows the effect ofifferent probe concentrations to the hybridization process. Whenhe probe’s concentration is 0–35 times as much as primer’s, ECLntensity has a wide range increasing. When the rate exceeds 35,he increasing becomes flattened. Taking both ECL signal intensity

Fig. 2. Probe concentration optimization. The probes were added to hybridizationsystem at concentrations of 5, 15, 25, 35, 45, and 60 times primer concentration(20 pmol).

and detection cost into account, we set the concentration of probeas 35 times as primer’s in the following experiments.

pH value is also a very important factor for nucleic acidhybridization. Suitable pH value can improve hybridization effi-ciency. As shown in Fig. 3, the variety of ECL intensity on the pHrange of 6.0–9.0 was studied. The ECL intensity increases with theincreasing pH from 6.0 to 7.0, and decreases from 7.0 to 9.0. There-fore, we selected pH 7.0 as the optimal pH value.

Other factors, such as annealing temperature and annealing timeare also crucial to this hybridization process. In our experiment, theTm of F. oxysporum f. sp. Cubense and X. oryzae pv. Oryzae was 50 ◦Cand 63 ◦C, respectively, the same as the annealing temperature inPCR programs. We incubated mixtures at their annealing tempera-ture for an hour, which was enough for the hybridization of nucleicacids.

In the process of ECL detection, temperature plays an important

Fig. 3. Hybridization efficiency under different pH. Hybridization running solution(pH 6.0–9.0, in 0.5 increment) was added to 20 �L PCR products, 10 �L biotin-probesand 10 �L TBR-probes.

432 J. Wei, B. Wu / Sensors and Actuators B 139 (2009) 429–434

Fig. 4. Sensitivity of this ECL-PCR method. Different amounts of F. oxysporum f.sw2F

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Fig. 5. ECL detection results of infected and uninfected samples. AAA, Musa AAA;

72.9 ± 17.67 cps, respectively. According to the threshold valueof 128.57 cps, the results show that banana samples of columns1–5 are all infected, and rice sample of column 6 is uninfected.

p. Cubense DNA were added to DNA extracted from 1 mg healthy banana leaves,hich are 320 ng/mg, 160 ng/mg, 80 ng/mg, 40 ng/mg, 20 ng/mg, 10 ng/mg, 5 ng/mg,

.5 ng/mg and 1.25 ng/mg, respectively. The unit ng/mg represents the amounts of. oxysporum f. sp. Cubense DNA in 1 mg healthy banana leaves.

The quantity of magnetic beads is highly important to the ECLetection. The biotin-labeled PCR products are linked to the sur-

ace of streptavidin-coupled beads though the highly selectiveiotin–streptavidin linkage [7,25]. The appropriate amount of mag-etic beads can capture the entire special PCR products. But if themount of magnetic is excess, the excessive beads will be absorbedn the surface of electrode, and hinder the reaction of TPA and TBR.o 10 �L beads are suitable to be added to each 20 �L hybridizationroduct by studying repeatedly.

.2. Sensitivity and reproducibility of this method

The sensitivity of this proposed method was estimated by detec-ion of F. oxysporum f. sp. Cubense DNA in a background of DNAxtracted from 1 mg healthy banana leaves. We added differentmounts from 1.25 ng to 320 ng of F. oxysporum f. sp. Cubense DNAo healthy banana leaves’ DNA, respectively, and used as DNA tem-lates in PCR amplification. As shown in Fig. 4, the sensitivity allowsetection of as little as 1.25 ng of F. oxysporum f. sp. Cubense DNA.he values of the relative standard deviation (RSD) are in the rangef 7.7–14.4% as shown in Table 2. With the decrease of F. oxysporum. sp. Cubense DNA concentration, the RSD is increasing slowly in ancceptable range.

.3. The ECL detection of plant pathogenic bacteria

Banana leaves including three different varieties of bananaamples (Musa AAA, Musa ABB, and Musa AAB) and rice sam-les were detected by the proposed method, as shown in Fig. 5.he blank control signals of Musa AAA, Musa ABB, Musa AABnd rice are 55.4 ± 14.90 cps, 54.03 ± 11.51 cps, 53.9 ± 8.46 cps and9.85 ± 16.24 cps, respectively. To define if a sample is plantathogenic bacteria-positive, a threshold value was calculatedased on the mean of blank control plus three times the stan-ard deviation [30]. According to the formula, the highest valuef rice’s blank control used as the threshold value for plantathogenic bacteria-positive was set at 128.57 cps. ECL signal

ess than 128.57 cps should not be indicated as plant pathogenicacteria-positive under the conditions. The results show that theignals of controls are under the threshold value, and the signalsf infected samples are much higher than threshold value, whichrove that the signal-to-noise ratio of ECL detection for infected

ABB, Musa ABB; AAB, Musa AAB; 10 �L biotin-probe and 10 �L TBR-probe wereadded to 20 �L PCR products. All these samples were detected under the optimizedconditions.

samples is great enough to identify whether the sample is infectedor not.

To further confirm the feasibility of the proposed method, sixsuspected samples were detected, as shown in Fig. 6(a). Columns1–5 are banana samples, and column 6 is rice sample. The sig-nals of these samples are 384.36 ± 24.65 cps, 330 ± 7.57 cps,427.74 ± 19.63 cps, 350.1 ± 16.53 cps, 379.51 ± 26.22 cps,

Fig. 6. (a) ECL detection results of suspected samples. Columns 1–5, banana sus-pected samples; column 6, rice suspected sample. (b) 1% agarose gel electrophoresisresults. Lanes 1–6 correspond to columns 1–6 in (a); M: marker.

J. Wei, B. Wu / Sensors and Actuators B 139 (2009) 429–434 433

Table 2Recovery of ECL-PCR detection.

Concentration (ng/mg) ECL intensity (cps) Standard deviation (SD) Relative standard deviation (RSD) (%)

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In order to verify the veracity of these ECL detecting results, weerformed agarose gel electrophoresis analysis for the same PCRroducts as that of ECL experiments. The gel was prepared with 1%f agarose containing 0.5 �L mL−1 of GoldViewTM as fluorescenceye. After electrophoresis, the PCR products were analyzed undern image analysis software (Quantity OneTM, Bio-RAD, CA, USA), ashown in Fig. 6(b). Lanes 1–6 correspond to 1–6 in Fig. 6(a). Lane

is represented for DNA marker. In lanes 1–5, bands between00 bp and 400 bp are observed, coincident with the 325 bp PCRroducts of F. oxysporum f. sp. Cubense’s ITS sequences, which dis-lay banana samples are infected. In lane 6, no band appears whichemonstrates this rice sample is uninfected. The results of gel elec-rophoresis are consistent with the results of ECL detection.

According to the target pathogenic bacteria, we can design aair of universal sequences, and introduce them to the 5′-terminalf sense primer and anti-sense primer, respectively, to realize ECL-CR detection of various target pathogenic bacteria using a pairf effective probes, TBR-probe and biotin-probe. So in addition todvantages of ECL-PCR methods, such as high sensitivity, simplicity,tability and free of stains, this proposed method for detection oflant pathogenic bacteria is cost-effective.

. Conclusion

With F. oxysporum f. sp. Cubense and X. oryzae pv. Oryzae ashe model plant pathogenic bacteria, a novel method for rapidetection of plant pathogenic bacteria has been developed. Sev-ral advantages of the proposed method should be highlighted.irst, streptavidin coated magnetic beads were used to separate andmmobilize the amplified PCR products of plant pathogenic bacteriahrough biotin-probe, indicating the magnetic beads-PCR productsan readily be collected on the electrode surface by using a mag-et and the electrode can be reused by simply washing out theeads from the surface. As a result, lead to the proposed methodor detection of pathogenic bacteria is highly sensitive, simple andapid. Second, the proposed method is potential as a universalethod for rapid detection of different plant pathogenic bacte-

ia by adding those universal sequences to the terminal of PCRrimers. As a result, this study can provide a feasible alternative toresently available pathogenic bacteria detection technique, as wells provides a feasible approach on developing ECL sensors to meethe demand of rapid detection of plant and animal bacteria, fungi,iruses, and other specific nucleic acid sequences. However therere still plenty of spaces for future explorations such as multiplexnd on-line detection of pathogenic bacteria using the proposedCL-PCR method, which are further pursuits of our efforts.

cknowledgements

This research is supported by the National Natural Science Foun-ation of China (30800261; 30870676; 30700155; 30600128), the

ational High Technology Research and Development Program ofhina (863 Program) (2007AA10Z204), the Natural Science Foun-ation of Guangdong Province (7005825), and the Learning androducing Combination of Guangdong Province and Educationepartment (2007B090400040).

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We are grateful to Yusheng Fang (College of Nature Resourcesand Environment, South China Agricultural University, Guangzhou,China) for kindly providing F. oxysporum f. sp. Cubense and LiexianZeng (Institute of Plant Protection, Academy of Agricultural Sci-ences, Guangzhou, China) for kindly providing X. oryzae pv. Oryzaeand infected rice leaf samples.

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4 Actua

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Baoyan Wu received a BS degree in agriculture from Tianjin Agricultural Collegein 1998, MS degree in 2004 and PhD degree in 2007 in zoology from Nankai Uni-

34 J. Wei, B. Wu / Sensors and

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Biographies

Jie Wei received a BS degree in biology science from Anhui University in 2006.She is currently working on a MS degree in optics at Institute of Laser Life Science,South China Normal University. Her research interests include specific nucleic acidssequences analysis and detection of pathogenic bacteria in plants and animals usingelectrochemiluminescence systems.

versity. She worked as a lecturer at South China Normal University in 2007. Herresearch interests include biosensor and applications of nanomaterials in biologicalsystems.