An improved electrochemiluminescence polymerase chain reaction

method for the detection of Fusarium wilts

Jie Wei, Xiao Ming Zhou *

MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University,

Guangzhou 510631, China

Received 16 January 2008

Abstract

An improved electrochemiluminescence polymerase chain reaction (ECL-PCR) method was developed and applied to detect

Fusarium wilt. Briefly, the internal transcribed spacer (ITS) sequence of Fusarium oxysporum f. sp Cubense (FOC) was amplified

by PCR. Two universal fragments, which were complimentary to Ru(bpy)32+ (TBR) labeled probe and Biotin labeled probe,

respectively, were connected to the tail of primers so that all the PCR products got universal sequences. Then biotin labeled probes

and TBR labeled probes were hybridized with the PCR products at the same time. Through the specific interaction between biotin

and streptavidin, the PCR products were captured by streptavidin coated magnetic bead and then detected by ECL assay. The

experiment results showed that the healthy banana samples and infected ones can be discriminated by this ECL-PCR method. This

improved ECL-PCR approach is useful in Fusarium wilt detection due to its high sensitivity, simplicity and stability.

# 2008 Xiao Ming Zhou. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: Electrochemiluminescence; Polymerase chain reaction; Fusarium wilts

FOC is the causal pathogen of wilt disease of banana. Fusarium wilts caused by FOC is one of the most destructive

diseases of banana worldwide. It is a classic vascular wilt disease in which the fungus gains entry to the water

conducting xylem vessels, then proliferates within the vessels causing water blockage. The typical symptoms include

wilting and death of the leaves, followed by death of the whole plant. A cost-effective measure of control for this

disease is still not available [1]. Recently, ECL-PCR techniques has been developed [2–5] due to its sensitivity, safety

and simplicity. This study developed an improved ECL-PCR technique for FOC detection. The principle of this ECL-

PCR method is showed in Fig. 1. The ITS (internal transcribed spacer) sequence of FOC was amplified by PCR. Two

universal fragments were added to the tail of primers so that all the PCR products contained these two universal

sequences. Then biotin labeled probes and TBR labeled probes, which were complimentary to the universal fragments,

respectively, were hybridized with the PCR products at the same time. After that, the PCR products were captured by

magnetic bead though the specific interaction between biotin and streptavidin and detected by ECL [6]. This is a highly

effective method to detect Fusarium wilt which is caused by FOC with high sensitivity, specificity and simple

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Chinese Chemical Letters 19 (2008) 959–961

* Corresponding author.

E-mail address: zhouxm@scnu.edu.cn (X.M. Zhou).

1001-8417/$ – see front matter # 2008 Xiao Ming Zhou. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

doi:10.1016/j.cclet.2008.05.033

operation procedures. As we connect universal sequence to the PCR primers, the cost of this ECL-PCR approach can

be significantly reduced.

1. Experimental

Race 4 Fusarium oxysporum f. Cubense (FOC) was obtained from College of Nature Resources and Environment,

South China Agricultural University (Guangzhou, China). b-Mercaptoethanol and cetyltrimethyl ammonium bromide

(CTAB) were purchased from AMRESCO, Netherlands. The streptavidin micro-beads were purchased from MACS,

Germany. TPAwas purchased from Aldrich Chemical Company, the Ru(bpy)32+ N-hydroxyl-succinimide ester (TBR-

NHS ester) was purchased from Sigma, USA. PCR primers and probes were all designed by our lab and synthesized by

Shanghai Sangong Biological Engineering & Technology Services Co. Ltd. (SSBE). The probes were labeled with

biotin by SSBE or TBR by our lab, respectively. The forward primer was 50-TAACTGAATAGACTAAGACAG-

CAAAATGCGATAAGTAAT-30. The reverse primer was 50-CTAATCAACGACCTTGTATCTATTCCTACCT-

GATCCGAGG-30. The TBR probe was 50-TBR-TAACTGAATAGACTAAGAC-30. The Biotin-probe was 50-biotin-GATACAAGGTCGTTGATTAG-30. The CTAB method for sample extraction and purification reported by

Schutzbank and Smith [7] was used in this study. PCR was conducted in a thermal circler (PTC—100, MJ Research

Inc., USA). The amplification protocol consisted of initial denaturation for 3 min, primer annealing for 1min and

extension for 1 min. The annealing temperature is 50 8C. After amplification, the biotin-probes and TBR-probes were

added to the PCR products to perform hybridization. The mixture was incubated for 5 min at 94 8C and 1 h at 50 8C in

the PCR system. Then, hybridization products were added to binding buffer. Streptavidin coated magnetic beads were

added. The mixture was then shaken at room temperature for 30 min. After washing and removing the supernatant, the

samples were added to the detection cell of ECL analyzer (built in our lab) [8]. Each sample was detected 4–5 times

and analyzed with statistical method.

2. Results and discussion

Fig. 2a shows the ECL detection results of both healthy and infected samples. All these samples were grinded with

sterile mortar and extracted with CTAB, precipitated, treated with chloroform, and precipitated with isopropanol to

obtain a purified DNA matrix. Column 1 is represented for healthy banana stem sample signals (negative control), the

value is 77.9 � 2.4 cps. Column 2–5 are the signals of infected banana stem sample, which were infected by our lab.

We inoculated race 4 F. oxysporum f. Cubense to the healthy banana stems and cultivated at 27 8C for 2–3 days. The

value of 2–5 is 424.6 � 20.1 cps, 436.3 � 14.2 cps, 427 � 18.4 cps, 443 � 31.8 cps, respectively. As the threshold

value can be derived as the mean of blank control plus 3 times the standard deviation (S.D.) of the background signal

[9], according to the data, we set the threshold as 85.1 cps. The signal to noise ratios of ECL detection for infected

samples is so great (>5) that we could confirm whether the samples have been infected or not. In order to verity the

feasibility of this method, 1% agarose gel electrophoresis analysis for PCR products was performed in the experiment

(Fig. 2b). The electrophoresis conditions are 1% agarose gel at 80 V for an hour. Lane B is represented for negative

control. Lane M is represented for markers, the six ladders was 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp,

respectively. Lane 1–4 corresponds to column 2–5 in Fig. 2a. Each lane 1–4 has a band between 300 and 400 bp which

J. Wei, X.M. Zhou / Chinese Chemical Letters 19 (2008) 959–961960

Fig. 1. The principle of this ECL-PCR method for detection of Fusarium wilts.

was consistent with the expected PCR products 325 bp. The results of gel electrophoresis are consistent with the

results of ECL detection.

In this study, an ECL-PCR method for detection of Fusarium wilt was described. The ITS sequence can be

amplified specifically by PCR and the universal sequences which were introduced to the primers can significantly

reduce the cost when we apply this method to detect several different pathogenic bacteria. The healthy samples and

infected ones can be discriminated with this method. Compared with the traditional detection methods, this novel

ECL-PCR is a safe, low background noise and specific technique. Due to its sensitivity, safety and simplicity, this

approach will have an enormous potential for plant pathogenic bacteria.

Acknowledgments

This research is supported by the National Natural Science Foundation of China (No. 30600128; 30470494) and the

Natural Science Foundation of Guangdong Province (No. 7005825; 7117865).

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Fig. 2. ECL detection results of both healthy and infected samples and the agarose gel electrophoresis analysis results of the PCR products.