Plant virus infection is a major factor in crop yield and has been responsible for causing severe losses in pepper crop production. Cucumber Mosaic Virus (CMV), a type species of the genus Cucumovirus is a prevalent plant pathogen all over the world and has the widest host range of over 885 plant species in 65 families (Palukaitis and García-Arenal, 2003). CMV causes economic damages to pepper production in Korea. Since the early 1980s, CMVP0 type isolates, such as CMV strain Fny, are the most detrimental virus in pepper farms in Korea. In the middle of the 1990s, pepper cultivars resistant to CMV-P0 isolates have been commercially produced by pepper breeders in Korea. However, the high resistance of pepper cultivars showing against CMV-P0 isolates was overcome by a new CMV-P1 type isolate (Lee et al., 2006). Recently, CMV-P1 isolates or related CMV isolates cause severe damage to pepper production in Korea and many Asian countries. CMV has a tripartite genome of positive-sense singlestranded RNAs, designated as 1, 2, and 3 in order of decreasing size (Peden and Symons, 1973). RNA2 codes for the 2a protein, which is a RNA-dependent RNA polymerase of the replication complex, whereasRNA1 codes for the 1a protein, another subunit of CMV replicase complex (Hayes and Buck, 1990; Palukaitis and García-Arenal, 2003). The 2b protein is expressed from a subgenomic RNA4A, derived from RNA2, and suppresses RNA silencing of host plants (Ding et al., 1994; Brigneti et al., 1989). RNA3 codes for two proteins, 3a and capsid protein (CP), which are involved in viral movement and encapsidation, respectively (Canto et al., 1997; Kaplan et al., 1997). Some isolates of CMV also contain RNA 5 or non-subgenomic RNA species that is a satellite RNA (Palukaitis et al., 1992). Immuno-assays have been widely applied in the diagnosis of plant viruses nation as screening methods due to their high sensitivity, specificity, fast detection speed and low-cost (Tang et al., 2009; Zhang et al., 2006) Various immune-assay methods have been reported for detection of CMV, such as enzyme-linked immunosorbent assay (Zein et al., 2006), immune-capture Reverse-transcription (RT)-PCR (Sharman et al., 2000), and Tissue-print combined with molecular detection (Chen, 2011). However, this kind of determination required some expensive equipment, for example, a microplate reader. 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 userfriendly 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 (Fu et al., 2007; Ding et al., 1999). The emerging research field of non-instrumental measurements of multiple residues provides the possibilities for simultaneous detection of CMV from pepper and other crops. As a rapid, on-site, easy and low-cost method, rapid immune-gold strip kit (RIGS) method plays a role in the determination of CMV. In the RIGS, colloidal goldnano- particles (AuNPs) for CMV 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 immune-assay, which named RIGS. Colloidal gold immune-assay has been developed and applied increasingly in various research fields such as for the detection of insectcides, toxins, hormones, animal viruses, and bacteria (Wang et al., 2005; Lai et al., 2008; Kranthi et al., 2009; Zheng et al., 201; Moon et al., 2012; Xu et al., 2012; Xin 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 CMV from a variety of horticultural and ornamental crops.
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 Watmann (USA). Filter paper, Endosulfan-diol, colloidal gold (20 nm), 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
CMV isolate Vch (CMV-Vch) from pepper was propagated in Nicotiana tabacum (cv. Samsun NN) after mechanical inoculation with CMV-Vch particles. For CMV purification, the systemic leaves of the inoculated N. tabacum plants were harvested post 14 days inoculation and the leaf tissues were used for CMV purification, as described previously (Peden and Symons, 1973). Polyclonal antibodies were produced in rabbits by immunization with the purified CMV-Vch 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 0.1 mg CMV 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 0.01 M, sodium phosphate buffer (pH 7.2), according to manufacturer’s instructions (PALL, USA).
Antibody Labeling
The affinity purified immunoglobulin (IgG) was conjugated to colloidal gold (40 nm) according to the methods, as described previously (Horisberger, 1989; Leuvering et al., 1980). 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 0.1 mg/ml in sodium phosphate buffer (0.001 M, pH 7.0). The pH of colloidalgold solution and the diluted IgG was adjusted to pH 6.0 to 8.0 with 0.1 M Na2CO3. Ten aliquots of variable concentrations (0.01-0.1 mg/ml) of the diluted IgG were prepared in 0.2 ml sodium phosphate buffer, and added separately to 1 ml of the colloidal-gold solution. 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 colloidal-gold solution (pH 6.0) followed by the addition of 10 ml of filtered 10% ovalbumin, pH 6.0 with gentle stirring for 20-25 min. 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 5 ml conjugate dilution buffer (0.01 M Tris, 3% bovine serum albumin, 1% sucrose and 0.1 M sodium azide) and stored at 4°C.
Preparation of RIGS Kit
A novel format (Fig. 1) was designed for RIGS kit, so that it enables the detection of CMV on a single strip. The colloidal-gold-conjugated solution of antisera raised against CMV 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. CMV IgG-bovine serum albumin conjugate was diluted in 0.02 M sodium phosphate buffer, (pH 7.4) containing 1% sucrose to a final concentration of 3 μg/mL and applied as a 0.5mm 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 CMV-IgG conjugate, which was applied to each membrane in 1g/L CMV 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. CMV Ig-ovalbumin conjugate was diluted in 0.02M sodium phosphate buffer (pH7.4) containing 1% sucrose to a final concentration of 3μg/mL and applied as a 1mm thick, 26 cm line, at 0.5cm 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 10-15min and washed twice with 0.01 M, sodium phosphate buffer, pH7.2 before drying it for 10-15min at 50°C. A polyethylene plastic sheet (26 × 8cm) 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 4 cm 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 2 mm on it. A filter pad was placed to overlap 2mm 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 coldlaminated 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 CMV-Infected Samples
Leaf tissues of pepper cultivars, cucumber, and melon plants infected by CMV were extracted with 5 mL of 0.5 × PBS (pH7.4) solution using tissue homogenizer (Agdia, USA) in a plastic bag. Then the crude sap in a plastic bag was briefly precipitated by hands and the CMV-RIGS strip was soaked in supernatant of the extract and incubated for 5 min. Reactivity and specificity of CMV-RIGS kit was done as describe above.
RESULTS and DISCUSSION
Polyclonal sera specific to CMV were produced from rabbit and the IgG specific to CMV was further purified using protein A affinity chromatography from sera. The concentration of CMV-IgG was 2.45 mg/mL. In the production of AuNPs conjugated with CMV-IgG, the particle size is inversely proportional to the sodium citrate volume. In the CMV-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 400 nm 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 CMV-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 evaluated, 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 CMV-IgG to AuNPs was 3 μg/mL (Fig. 2). 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 0.1M NaCl added to the AuNPs solution was in the range of 1-10 μL/mL for CMV to determine the optimal pH of CMV IgG for the stable color detection with AuNPs. Transmission electron microscopy showed that AuNPs conjugated with CMV-IgG were evenly distributed at pH6.0 (Fig. 3). The results suggest that 3 μg CMV-IgG is sufficient for 1mL of AuNPs and the optimal conjugation between CMV-IgG and AuNP is done at pH6.0.
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 CMV and analytes. In addition, the blocking buffers with the BSA or OVA were evaluated to get the optimal blocking buffer. The results show that 0.01M PBS (pH7.4) containing 2% ovalbumin, 2% sucrose and 0.02%NaN3 was chosen as the optimal blocking buffer for the conjugate pads.
The competitive RIGS strip for CMV detection was defined as the lowest CMV concentration producing the color on the test line significantly weaker than that of the negative control strip. A serial end-point dilution of purified CMV virions was used for the sensitivity of the RIGS. The sample pad of RIGS strip was soaked in aliquot of crude sap, showing that the color intensity was readily shown after 5 min. The intensity of the test lines decreased with the dilution of CMV virion concentration. It is clearly shown that CMV can be successful detected as amount as 0.0156 mg/L using the developed RIGS kit (Fig. 4). The reliability of the RIGS strip was evaluated by performing the CMV detection in pepper as well as other host species infected by CMV or mock (healthy). Methods for extraction of CMV-infected plants were the same as described before. CMV infection were successfully detected from pepper cultivars, cucumber and melon plants infected by CMV (Fig. 5(a)), suggesting that the RIGS is useful for CMV detection from economically important crops, such as pepper (Choi et al., 2001; Choi et al., 2005). To determine whether the CMV-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(b), the RIGS strips did not cross-react to any viruses unrelated to CMV, confirming high specificity and reliability of the RIGS specificity for CMV detection from pepper and other crops (Cho et al., 2007). The stability of the RIGS strip assay was evaluated by comparing the analysis of CMV-infected crude sap before and after the strip storage. 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. In terms of diagnosis expense, the CMV-RIGS kit is 5-fold cheaper than other diagnosis methods, such as RT-PCR analysis, though high quality of antibody production is required for the CMVRIGS kit. Taken together, CMV-RIGS kit is a rapid, userfriendly and simple immune-chromatographic dipstick and the kit is suitable for on-site determination of suspect CMV-infected plants as well as for laboratory diagnosis.
적 요
‘급속면역금나노입자막대 (RIGS) 키트’라 명명된 빠르며 사 용자 편의 및 간단한 면역흡착막대 방식의 키트가 오이모자이 크바이러스 (CMV)의 현장진단을 하기 위하여 개발되었다. 토 끼의 CMV항혈청에서 정제된 면역글로블린G (IgG)는 단백질 A 크로마토그래피법으로 정제되었으며 이후 금나노입자와 결 합하여 니트로셀룰로스막에서 진단선 표시하도록 고안되었다. CMV항체와 비특이적으로 결합하는 단백질A를 같은 진단막 대에서 대조선으로 이용되었다. RIGS-CMV 키트를 이용한 진 단은 의심 식물 시료를 완충액이 들어간 플라스틱 봉지에 넣 은 후 착즙 후 진단막대기를 넣으면 된다. 결과는 5-10분 후 알 수 있다. CMV가 감염된 고추, 오이 및 멜론의 즙액에 RIGS 막대기를 넣고 진단한 결과 CMV 농도에 비례하여 진 단선이 형성됨이 관찰되었으며, 이들 키트들은 CMV와 연관 되지 않은 다른 고추바이러스들에서 비특이적 반응이 형성되 지 않았다. 이런 결과들은 RIGS-CMV 키트가 매우 민감하며, 진단에 별 다른 실험실 기술이나 경험이 필요하지 않는 다는 것을 의미한다. 그러므로, RIGS-CMV 키트는 CMV 감염이 의심되는 식물체들의 현장 진단 뿐만 아니라 실험실에서의 CMV 진단에 효과적이다.
