Plant virus infection is a major factor in crop yield and has been responsible for causing severe losses in production of horticultural and ornamental crops, such as tomato. Tomato bushy stunt virus (TBSV) is the type member of the Tombusviridae family consisting of several distinct but closely related genera of spherical RNA plant viruses. TBSV is a 30-nm icosahedral virus with singlestranded, positive-sense RNA genome of approximately 4.8kb that encodes five open reading frames (ORFs) (Hearne et al., 1990). The 5' proximal ORF (p33) and its readthrough product (p92) encode the viral RNA-dependent RNA polymerase (Oster et al., 1998). The coat protein ORF (called ORF2) lies in the middle of the genome and is translated from a subgenomic RNA (sgRNA 1). The CP is required for viral encapsidation and systemic spread in Nicotiana benthamiana (Qu and Morris, 2002). A smaller sgRNA (called sgRNA2), derived from 3' fourth of the genome, is responsible for the expression of two nested genes (ORFs 3 and 4). The ORF3 encodes p22 involved in the viral cell-to-cell movement and as well as systemic movement in the infected plant (Scholthof et al., 1993, 1995). The ORF4 encodes p19 involved in the suppression of RNA silencing. P19 is shown to be necessary for systemic movement in some host species, but not required for cell-to-cell movement (Chu et al., 2000; Scholthof et al., 1995).

TBSV was first isolated from tomato with viral symptoms in UK, and subsequently reported in South America, USA, Morocco, Portugal, Tunisia and Japan (Fischer and Lockhart, 1977; Koenig and Avgelis, 1983; Pontis et al., 1968; Smith,1935). TBSV is readily transmitted by mechanical inoculation to a wide range of host plants. Natural transmission of the virus is occurred mainly through seed and soil, but it is not clear whether the transmission of the virus is done by insect vectors. Symptoms induced by TBSV in tomato plants include stunting and bushy growth, deformation and chlorotic spots on young leaves, and purpling and necrosis of older leaves. Severe yield losses associated with TBSV have been reported in peanut, tobacco, tomato, pepper and potato as well as in some ornamental crops (Martelli et al., 2001). Yields are reduced, and fruits are smaller and show chlorotic rings and blotches that lower the epidemic value of the crop (Gerik et al., 1990). TBSV caused epidemic outbreaks in tomato and eggplant crops in southern Spain and has spreading with the expansion of nipple fruit cultivation in Japan (Luis-Arteaga et al., 1996). Based on serological relationship and sequence identity, Petunia asteroid mosaic virus and Artichoke mottled crinkle virus are considered as TBSV strains (Luis-Arteaga et al., 1996). In Korea, a number of viruses including Cucumber mosaic virus, Tomato mosaic virus, Tobacco mosaic virus, Pepper mottle virus and Potato virus Y (PVY) were identified from tomato plants (Choi et al., 2010). Local outbreak of TBSV was observed from tomato showing chlorotic spots, malformation and necrosis on leaves, and chlorotic blotching, rings, and necrosis on fruits in Sacheon, Gyeongsangnam- do, Korea in 2007 (Kim et al., 2007). As colloidal gold has been widely used in immunoassay for large molecular, the nano-colloidal gold particles could replace the enzyme to be labeled to antibody in detection of plant viruses, such as commercial kits from Agdia corporation. Compared with enzyme immunoassay, the determination by colloidal gold-based immunoassay can be completed rapidly in one step. When the antibody labeled with colloidal gold particles is combined with the corresponding antigen, the colored immune-reactant can be visually detected. This user-friendly format possesses several advantages, such as a very short time for obtaining test results, long-term stability over a wide range of climates and relative in expense. These characteristics make it ideally suited for on-site testing by untrained personnel.

Various immune-assay methods have been reported for detection of TBSV, such as enzyme-linked immunosorbent assay, RT-PCR, and real-time RT-PCR (Kim et al., 2007). However, this kind of determination required some expensive equipment, for example, a microplate reader. The emerging research field of non-instrumental measurements of multiple residues provides the possibilities for simultaneous detection of TBSV from pepper and other crops. As a rapid, on-site, easy and low-cost method, rapid immunegold strip kit (RIGS) method plays a role in the determination of TBSV. In the RIGS, colloidal gold-nano-particles (AuNPs) for TBSV detection is commonly used as signal material. AuNPs are visible and can be detected with naked eyes. Therefore, the RIGS combined with AuNPs has been widely used. The goal of this study was to develop a rapid and simple detection method that was based on one step membrane-based competitive colloidal gold immunoassay, which named RIGS. Colloidal gold immunoassay has been developed and applied increasingly in various research fields such as for the detection of insecticides, toxins, hormones, animal viruses, and bacteria (Wang et al., 2005; Zein et al., 2006; Zhang et al., 2006; Kranthi et al., 2009; Lai et al., 2009; Moon et al., 2012; Xu et al., 2012; Li et al., 2013). In this paper, we describe the development of one-step colloidal gold-based assays (named RIGS combined with AuNPs) for the detection of TBSV from tomato.

MATERIALS AND METHODS

Chemicals

Nitro-cellulose membranes were obtained from Millipore (USA). Semi-rigid polyethylene sheets, sample wick, absorbent pad used in the development of immuno-chromatographic, and adhesive tape were purchased from Whatman® (USA). Filter paper, Endosulfan-diol, colloidal gold (40nm), ovalbumin, protein A, Freund’s complete and incomplete adjutants, analytical grade buffer chemicals, and all other reagents used in RIGS production and protein conjugation were purchased from Sigma-Aldrich (USA).

Antibody production

A strain of TBSV (TBSV-B) was isolated from tomato showing fruit decoloring , mosaic and leaf deformation in a greenhouse, Busan, 2008 (Fig. 1(a) and (b)). TBSV-B was propagated in Nicotiana clevelandii after mechanical inoculation with tomato leaf sap infected by TBSV-B. For TBSV purification, the systemic leaves of the inoculated N. clevelandii were harvested post 14 days inoculation and the leaf tissues were used for TBSV purification, as described previously (Martelli et al., 2001; Kim et al., 2007). The purified TBSV-B particles were verified using trans mission electron microscopy, according to standard protocol (Fig. 1(c); Sambrook et al., 1989). Polyclonal antibodies were produced in rabbits by immunization with the purified TBSV-B conjugates. For the first immunization, the immunogen (0.1 mg) was suspended in 2 ml of 0.5X phosphate buffer saline (PBS), emulsified with 2 ml Freund’s complete adjuvant and injected subcutaneously in two rabbits for each immunogen. Four booster dose immunizations were given at monthly intervals after the first immunization using 0.1mg TBSV virions in 2 ml of 0.5X PBS buffer emulsified with Freund’s incomplete adjuvant. About 10 - 12 ml serum was collected from the marginal ear vein, 14 days after each booster. Sera that exhibited a strong immunogenic response were further purified by affinity chromatography using Protein A affinity chromatography columns and dialyzed against 0.01M, sodium phosphate buffer (pH7.2), according to manufacturer’s instructions (PALL, USA).

Antibody labeling

The affinity purified immunoglobulin (IgG) was conjugated to colloidal gold (40nm) according to the methods, as described previously (Horisberger, 1989). The optimal concentration of antibody for conjugation with colloidal gold was determined by titrating aliquots of diluted IgG with colloidal gold. The purified IgG was diluted to a concentration of 0.1mg/ml in sodium phosphate buffer (0.001M, pH7.0). The pH of colloidal gold solution and the diluted IgG was adjusted to pH8.0 with 0.1M Na₂CO₃. Ten aliquots of variable concentrations (0.01 - 0.1mg/ml) of the diluted IgG were prepared in 0.2 ml sodium phosphate buffer, and added separately to 1 ml of the colloidalgold solution, as described previously (Li et al., 2013). After incubating the mixture for 10 min, 0.1 ml of 10% NaCl was added to the tubes and the absorbance was measured at 520 nm. The least amount of protein required to stabilize the colloidal gold was identified from the abscissa in the curve drawn from the concentration and the absorbance. Aliquot (approximately 10 ml) of purified IgG (0.1 mg/ml) was added drop-wise to l00 ml of colloidalgold solution (pH8.0) followed by the addition of 10ml of filtered 10% ovalbumin, pH8.0 with gentle stirring for 20- 25min. The solution was incubated for 1 h at 4 °C and centrifuged at 15,000g for 30 min at 4°C. The supernatant was discarded and the loose precipitate of gold conjugate was re-suspended in 5ml conjugate dilution buffer (0.01M Tris, 3% ovalbumin, 2% sucrose, 0.2% NaCl and 0.03% sodium azide) and stored at 4°C, as described previously (Lai et al., 2010).

Preparation of RIGS kit

A novel format (Fig. 2) was designed for RIGS kit, so that it enables the detection of TBSV on a single strip. The colloidal-gold-conjugated solution of antisera raised against TBSV was mixed in equal quantities and applied on 26 × 1.7 cm conjugate pads (Standard 14) and dried under dry air for 10 - 15 min. TBSV IgG–ovalbumin conjugate was diluted in 0.02 M sodium phosphate buffer, (pH8.0) containing 1% sucrose to a final concentration of 2 mg/mL and applied as a 0.5 mm thick, 26 cm line, centrally at 1.25 cm from the top and bottom ends on one side of a 2.5 × 26 cm nitrocellulose plastic backed membrane strip, using a locally fabricated airbrush device (Innovative Biosciences, India). Nitrocellulose membrane was cut into sections (2.5 cm × 26 cm). Test line was coated with TBSV-IgG conjugate, which was applied to each membrane in 1 g/L TBSV using TLC conjugate sampler (Sambrook et al., 1989). The distance between the test line and control line was 6 mm. The test strips were dried at 37°C for 30 min. TBSV IgG–ovalbumin conjugate was diluted in 0.02 M sodium phosphate buffer (pH8.0) containing 1% sucrose to a final concentration of 3 μg/mL and applied as a 1mm thick, 26 cm line, at 0.5 cm from the top end of the membrane. The membrane was dried at 50°C under a dry wind blower for 10 - 15 min and blocked with PBS containing 2% ovalbumin and 1.5% sucrose. The membrane was dried at 50°C under a dry wind blower for 10-15 min and washed twice with 0.01 M, sodium phosphate buffer, pH7.2 before drying it for 10 - 15 min at 50°C. A polyethylene plastic sheet (26 × 8 cm) of 0.2 mm thickness was coated with acrylic adhesive on one side and the 2.5 cm wide membrane was placed centrally at a spacing of 1.5 cm from the top and 4cm from the bottom end of the sheet. The conjugate coated glass-fiber pad was placed on the lower end of the membrane, so as to overlap 2mm on it. A filter pad was placed to overlap 2 mm on the lower end of the conjugate release pad to act as sample pad and another pad (CF4) was placed to overlap 2 mm on the upper end of the membrane to act as absorbent pad. The assembly was cold-laminated using an 8 cm wide transparent adhesive tape. The laminated 26 × 8 cm assembly was cut into lateral- flow strips of 8 × 0.4 cm. The strips were stored in an airtight plastic bottle containing a desiccant pack.

Extraction of TBSV-infected tomato

Tomato leaf tissues infected by TBSV were extracted with 5 mL of 0.5X PBS (pH7.4) solution using tissue grinder (Agdia, USA). Then the crude sap was precipitated briefly and the supernatant was used for specificity of RIGS kit.

RESULTS AND DISCUSSION

TBSV-B was successfully purified from leaf tissues of N. clevelandii, showing denatured capsid proteins of TSWV-KP were detected on SDS-PAGE gel (Fig. 1(c) and data not shown). The purified TBSV-B particle is about 35 nm. Polyclonal sera specific to TBSV were produced from rabbit and the IgG specific to TBSV was further purified using Protein A affinity chromatography from sera. The concentration of TBSV-IgG was 1.0 mg/mL. In the production of AuNPs conjugated with TBSV-IgG, the particle size is inversely proportional to the sodium citrate volume. In the TBSV-IgG antibody-AuNP conjugation, the antibody was absorbed on the AuNP surface. The effects of pH values on the conjugation were studied by evaluating the absorbance between 400nm and 650 nm. Also, the main purpose of the assay was to allow visual evaluation, so it was only used as a qualitative assay to detect contamination at a threshold level. As the Na₂CO₃ concentration increased, the maximum absorption wavelength increased up to the optimal concentration and then decreased (data not shown). The definition of the optimal concentration of TBSV-antibody was the one that gave the required visibility and the best sensitivity. During antibody concentration optimization, the minimal stable polyclonal IgG concentration form antibody-AuNP conjugation was firstly designed and evaluated (Fig. 2), and then the optimal concentration was studied. Optimal immune-reagent concentration was selected as a clear color appearing in the negative control with the shortest time, and comparison of the intensity of color among samples and control could be easily distinguished by eye. The optimal concentration for polyclonal TBSV-IgG to AuNPs was 7 μg/mL (Fig. 3(a)). It is known that the amount of antibody and conjugates should be keep low enough to achieve good sensitivity, but it should be sufficient to provide an acceptable signal (Xu et al., 2012). Next, the amount of 0.1 M NaCl added to the AuNPs solution was in the range of 1-10μL/mL for TBSV to determine the optimal pH of TBSV IgG for the stable color detection with AuNPs. Transmission electron microscopy showed that AuNPs conjugated with TBSVIgG were evenly distributed at pH8.0 (Fig. 3(b)). The results suggest that 7 μg of TBSV-IgG is sufficient for 1mL of AuNPs and the optimal conjugation between TBSV-IgG and AuNPs is done at pH8.0.

In general, the analytical performance of the RIGS strip is affected by many parameters, such as the type and pore size of the membrane, blocking buffer and immunereagent amount. In this work, blocking buffers for the conjugate and sample pads were evaluated to study its effect on polyclonal IgG specific to TBSV and analytes. In addition, the blocking buffers with the BSA or ovalbumin were evaluated to get the optimal blocking buffer. The results show that 0.01M PBS (pH8.0) containing 2% ovalbumin, 2% sucrose and 0.03% NaN₃ was chosen as the optimal blocking buffer for the conjugate pads.

The competitive RIGS strip for TBSV detection was defined as the lowest TBSV concentration producing the color on the test line significantly weaker than that of the negative control strip. A serial end-point dilution of crude saps from TBSV-infected tomato leaves was used for the sensitivity of the RIGS. The sample pad of RIGS strip was soaked in aliquot of crude saps, showing that the color intensity was readily shown after 5 min (Fig. 4). The intensity of the test lines decreased with the dilution of TBSV in the infected tomato leaves. It is clearly shown that TBSV in plant tissues can be successful detected as amount as 7.81 × 10^–4 g/mL of tomato tissue using the developed RIGS kit (Fig. 4). The reliability of the RIGS strip was evaluated by performing the TBSV detection in pepper as well as other host species infected by TBSV or mock (healthy). Methods for extraction of TBSV-infected plants were the same as described before. TBSV infection were successfully detected from tomato plants infected by an isolate TBSV in a greenhouse, Jinju, Gyeonsangnam-do (Fig. 4 and data not shown), suggesting that the RIGS is useful for TBSV detection from economically important crops, such as pepper and tomato (Kim et al., 2007; Choi et al., 2010). To determine whether the TBSV-RIGS strip reacts with unrelated plant viruses, representative viruses infecting pepper cultivars were evaluated for specificity of the RIGS strips. As shown in Fig. 5, the RIGS strips did not cross-react to Tomato spotted wilt virus, PVY, Tomato yellow leaf curl virus that cause severe damage to tomato production. These results confirm the high specificity and reliability of the RIGS for TBSV detection in tomato and other crops (Fig. 5). The stability of the RIGS strip assay was evaluated by comparing the analysis of TBSVinfected crude sap before and after the strip storage (Daughtrey, 1996). The strips prepared from the same batch were stored at 4°C under dry conditions. After 6 months of the storage of the strips, color intensity and detection sensitivity did not show significant differences from those using the fresh strips (data not shown), indicating that the RIGS strip assay was highly stable at the room temperature conditions.

적 요

‘급속면역금나노입자막대 (RIGS) 키트’라 명명된 RIGS 키 트는 간단한 면역 흡착막대 방식으로 사용자가 빠르고 편리하 게 이용할 수 있으며 토마토덤불위축바이러스 (TBSV)에 대한 현장진단을 하기 위하여 개발되었다. 토끼의 TBSV 항 혈청에 서 정제된 면역글로블린G (IgG)는 단백질A 크로마토그래피 법으로 정제되었으며 이후 금 나노입자와 결합하여 니트로셀 룰로스막에서 진단 선을 표시하도록 고안되었다. TBSV 항체 와 비특이적으로 결합하는 단백질A를 같은 진단막대에서 대 조선으로 이용되었다. RIGS-TBSV 키트를 이용한 진단은 의 심 식물 시료를 완충액이 들어간 플라스틱 봉지에 넣고 착즙 후 진단막대기를 넣으면 5-10분 후 결과를 알 수 있도록 고안 되었다. TBSV가 감염된 토마토 즙액에 RIGS 막대기를 넣고 진단한 결과 TBSV 농도에 비례하여 진단 선이 형성됨이 관 찰되었으며, 이들 키트들은 TBSV와 연관되지 않은 다른 고추 바이러스들에서는 비특이적 반응이 형성되지 않았다. 이런 결 과들은 RIGS-TBSV 키트가 TBSV 진단에 다른 어려운 실험 이나 기술이 필요하지 않고 쉽게 병원균을 진단 할 수 있다는 것을 의미한다. 그러므로, RIGS-44 TBSV 키트는 TBSV 감 염이 의심되는 식물체들의 현장 진단 뿐만 아니라 실험실에서 의 TBSV 진단에 효과적이라 할 수 있다.