Yellow Virus Research Articles

Development and validation of protocols for product stewardship in transgenic white clover (Trifolium repens L.): detection of the AMV CP and npt2 transgenes in seeds, herbage and hay

White clover (Trifolium repens L.) is an important pasture legume in temperate areas throughout the world, providing fodder for grazing animals and improving soil fertility via symbiotic nitrogen fixation. However, the persistence and stress tolerance of white clover is affected by several viruses, chiefly Alfalfa mosaic virus (AMV), Clover yellow vein virus (ClYVV) and White clover mosaic virus (WClMV). Efforts to introgress natural forms of virus resistance from other Trifolium spp. into white clover and lucerne (alfalfa) have had only limited success. This has been addressed by developing white clover germplasm exhibiting viral-coat-protein-mediated resistance to AMV and non-transgenic resistance to ClYVV. This report describes PCR-based assays for detecting the transgenes associated with the H6 transformation event in seeds, fresh leaves, air-dried leaves and mixtures of air-dried herbage of white clover and perennial ryegrass (hay). Although further development is required to convert these assays for use in the field, this paper demonstrates the ability to detect these transgenes in a range of agricultural products associated with the commercial use of white clover.

Geminivirus Modified Antioxidant Systems and Proline Accumulation in Tomato Plant

This study is conducted to examine the effects of Tomato yellow leaf curl virus (TYLCV) geminivirus infection on nitrogenous constituents, proline, nitrate reductase, phenylalanine ammonia lyase activity, total phenol content, glutathione, lipid peroxidation (MAD) and also activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.7) and catalase (CAT; EC 1.11.1.6) were estimated in both healthy and infected tomato plants. Infected plants showed reduction in total protein and nitrate reductase. Besides these, TYLCV infection caused an increase of total-N, amino-N, proline content in leaves, stems and roots, and also increase of phenylalanine ammonia lyase activity, total phenol content, glutathione, lipid peroxidation and activities of antioxidant enzymes in leaves were detected compared with healthy plants.

Molecular analysis of the 3' terminal region of Onion yellow dwarf virus from onion in southern Italy

Onion yellow dwarf virus (OYDV) is an economically important pathogen causing severe disease in garlic, onion and other Allium crops. Eleven isolates of OYDV, all from onion originating from Calabria, southern Italy, were genetically analyzed. An OYDV onion isolate from Sudan was also included in this study. The 3’ terminal region of about 2.5 kb of the twelve isolates were sequenced and the sequences comprising a part of the nuclear inclusion a (NIa-Pro), the complete nuclear inclusion b (NIb) and coat protein (CP) genes and the 3’ untranslated region (3’UTR), were compared to each other and to corresponding sequences of other OYDV isolates from different countries and Allium hosts. The within-population nucleotide identity of the Italian OYDV onion isolates was very high (more than 99.3%), whereas nucleotide identity between them and OYDV onion isolates from Germany was 94%, Argentina 92% and Sudan 87%. Recombination analysis among the complete 3’ terminal sequences showed putative recombination breakpoints in the NIb region of the Argentine isolate, with the minor parent related to the Sudanese isolate. Comparison between OYDV isolates from onion and isolates from garlic produced identities of 77-78% for the complete nucleotide region. When the 3’ terminal nucleotide sequence and the complete NIb protein were analyzed, the phylogenetic analysis generated rooted trees with high bootstrap values (100%), showing a genetic grouping into two well separated clades distinctive for onion and garlic isolates of OYDV. Phylogenetic analysis of CP protein and 3’UTR showed lower bootstrap separation values and no distinct sub-grouping of the OYDV isolates from the two major Allium species.

Thrips tabaci(Thysanoptera: Thripidae) andIris yellow spot virusAssociated with Onion Transplants, Onion Volunteers, and Weeds in Colorado

Abstract. Infestation by onion thrips, Thrips tabaci Lindeman, was determined on transplants of onion (Allium cepa L.) received in Colorado during March and April from out-of-state sources (Imperial Valley, CA; near Phoenix, AZ; and southern Texas) during 2004 to 2008. In the 5 years of the study, 50 to 100% of the transplant lots sampled arrived infested with thrips. Among infested transplant lots, the overall number of thrips averaged 0.15 to 0.63 per plant, with as many as four per plant in some lots. T. tabaci was the dominant thrips species in all seasons and locations of transplant origin. In addition, 19 of 83 (23%) tested lots had plants positive for Iris yellow spot virus. Iris yellow spot virus and T. tabaci were detected in volunteer onion plants as early as 1 May, a few weeks after the summer onion crop was planted, suggesting a possible role of infected volunteer plants in perennation of the virus between onion crops. Iris yellow spot virus and T. tabaci were detected in many common weeds inc...

FIRST REPORT OF AGERATUM YELLOW VEIN VIRUS AND PAPAYA LEAF CURL GUANGDONG VIRUS ON EUPHORBIA PULCHERRIMA IN CHINA

Three Euphorbia pulcherrima (poinsettia, family Euphorbia) plants showing leaf curl and vein thickening symptoms were collected in Fujian Province, China, in 2006. Total DNA was extracted from each sample using a CTAB method. A fragment of approximately 500 bp was amplified by PCR in each sample, using the special degenerate primer pair PA/PB (Deng et al., 1994). PCR products were gel-purified, ligated into pMD18-T vector (Takara Biotechnology, China), and sequenced. Six clones from each sample were sequenced. Phylogenetic analysis of the 500 bp fragments showed that these sequences formed three distinct branches, and sequence alignment among each branches (63.3% to 73.6%) in one sample indicated that each sample might be mixed infected by three distinct viruses. FE01, FE02 and FE03 isolates representing each virus were further studied. To amplify the full-length DNA-A of the three isolates, three pairs of abutting primers (FE01-F 5’-CCTTAGCAAGTAGTTCATTCCG-3’/FE01-R 5’-GACATGTCTTTGTCAGTTAGTGG-3’, FE02-F 5’-CCACTCAGAACGCTCCCTCA-3’/FE02-R 5’-GTTCGTGGTAGGGACCACTT-3’, and FE03-F 5’-TGCGCGCTCATCGCTTAGT-3’/FE03-R 5’-ATTATATTGGTCGAGGGCCCAC-3’) were designed based on the obtained sequences, respectively. They were determined to be 2751 (FJ487911), 2754 (FJ495183) and 2733 (FJ495184) nucleotides, and had the typical genome organization of Old World monopartite begomoviruses, respectively. Sequence comparisons revealed that the three isolates were most closely related to those of Euphorbia leaf curl virus (ELCV, AJ558121), Ageratum yellow vein virus (AYVV, FJ869908) and Papaya leaf curl Guangdong virus (PaLCuGDV, FJ869907), with 92.8%, 99.8% and 99.1% sequence identity, respectively. The attempt to detect a DNA-B or a betasatellite component by using specific primers (Briddon et al., 2002; Rojas et al., 1993) was unsuccessful. To the best of our knowledge, this is the first report of AYVV and PaLCuGDV and mixed infection of three begomoviruses in poinsettia in China.

FIRST REPORT OF LEEK YELLOW STRIPE VIRUS IN FOREIGN AND POLISH GARLIC PLANTS IN CENTRAL POLAND

Leek yellow stripe virus (LYSV), genus Potyvirus, family Potyviridae (King et al., 2011) is the most common and important virus infecting a wide range of Allium worldwide. The aim of this study was to detect and identify LYSV in leek and garlic plants originating in central Poland, and also materials from Belgium, Egypt, and Spain purchased in Polish markets in April 2014. Randomly collected 178 samples were tested by a double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA), according to the manufacturer's instructions (DSMZ, Braunschweig, Germany). All leek plants tested negative for LYSV, whereas 31 of 120 garlic bulbs tested positive. The presence of LYSV was confirmed by reverse transcription (RT)-PCR using total RNA extracted with the silica capture method (Boom et al., 1990; Malinowski, 1997) and primers 1-LYSV/2-LYSV (Parrano et al., 2012) designed to amplify a part of the N-terminal domain of the coat protein (CP) gene of the virus (363 bases). A sequence of the partial CP genes of the 12 LYSV isolates was submitted to GenBank (Accession Nos KM032272-KM032283). BLAST analysis of Polish sequences showed 96-99% identity at the nucleotide and amino acid levels. Sequences of Egyptian isolates, first representatives from this locations, showed 92 and 95% nucleotide and amino acid identities, respectively. However Spanish isolates revealed 95% and 97% nucleotide and amino acid identities, respectively. To the best of our knowledge, this is the first report of LYSV in foreign and Polish garlic plants available for purchase in central Poland. The accurate identification of viruses present in garlic plants, especially in imported plant material, will help to use the appropriate strategies to reduce viral incidence in garlic-growing areas.

FIRST REPORT OF AGERATUM YELLOW VEIN VIRUS ASSOCIATED WITH A NEW BETASATELLITE INFECTING CARICA PAPAYA IN CHINA

A Carica papaya plant exhibiting downward curling of the leaves and yellow vein was observednear Sanya (Hainan Province, China). Total DNA was extracted from three symptomatic samples (P1, P2 and P3) for amplifying part of begomovirus DNA-A and DNA-B using the primer pair PA/PB and PBL1v2040/PCRc1 (Zhou et al., 2001; Rojas et al., 1993). No DNA-B was detected but a ca. 500 bp DNA-A fragment amplified from all samples was cloned, sequenced and shown to have 100% nucleotide (nt) identity. The primer pair BE1 (5’-GGTACGCCGACGGCTGAACTTC-3’) and BE2 (5’-CGTACCTTGGATGCGGGAGTTGAAATG-3’) was designed to amplify the full-length DNA-A from P3, which was determined to be 2,759 nt in length (GenBank accession No. KM051844) and showed a nt sequence identity (99%) with Ageratum yellow vein virus (AYVV) from Lycopersicon esculentum (KC810890). When alpha- and betasatellite molecules were looked for using the universal primer pairs DNA101/102, UN101/102 and Beta01/02 by PCR, respectively (Briddon et al., 2002; Bull et al., 2003), no alphasatellite was detected, but a betasatellite amplicon of ca. 1.3 kb was obtained from the three samples. The complete betasatellite sequence from P2 (1,350 nt, GenBank accession No. KJ642219), shares the highest nucleotide sequence identity (66.6%) with Tomato leaf curl China betasatellite (GenBank accession No. AJ704617), and appears to be a new satellite species, for whichthe name Papaya leaf curl China betasatellite [China: Hainan: 2014] is proposed. To our knowledge, this is the first report of AYVV associated with a new betasatellite infecting papaya and their first identification in China.

Impact of soil salinity on fungal vector of rhizomania virus infecting beta vulgaris

Polymyxa betae Keskin is the only natural transmitting vector of the Beet necrotic yellow vein virus (BNYVV) among the cultivated sugar beet. This work aims to study the impact of salt stress on fungus-virus-host relationships. The fungal infected fine roots of sugar beet plant naturally infected with BNYVV were collected and treated with different salt concentrations (0, 2000, 4000, 6000 and 8000 ppm) of NaCl for one day prior to mixing it with a sterile soil. After virus symptoms appeared on leaves, disease severity has been determined, leave and roots tissues were collected for BNYVV detection. It was found that severe symptoms on sugar beet inoculated with treated roots by low salt concentrations (2000 and 4000 ppm) while at high salt concentrations, 8000 ppm less injury approximately as control treated with H2O. On the other hand it was found the cystosorial colonization was increased in low salt concentrations (2000 and 4000 ppm) while decrease in high salt concentrations (6000 and 8000 ppm) especially in 8000 ppm. The same trend results were observed in virus concentration in roots where as the BNYVV concentration was increased in low salt concentrations (2000 and 4000 ppm) while decrease in high salt concentrations (6000 and 8000 ppm). So the relation between capacity of fungal vector to infect the plant by virus and salinity concentration are antagonistic. Soil salinity extremes most often lead to decrease infection of BNYVV via effect on fungal zoospore. Key words: Polymyxa betae, sugar beet, rhizomania, beet necrotic yellow vein virus (BNYVV), salinity.

Ecogenomics of Geminivirus from India and neighbor countries: An in silico analysis of recombination phenomenon.

Recombination is one of the keys factor in evolutionary processes, involved in shaping the architecture of genomes and consequent phenotype. Understanding the recombination phenomenon especially among viruses will help in disease management. The present study aimed for in-silico analysis of recombination phenomenon among Begomoviruses. Particularly emphasizing on viruses strains reported from India and neighboring countries. A total of 956 virus sequences have been used in this study. The Tomato yellow leaf curl China viruses, namely gi|29825986|; gi|283468151|; gi|190559151| and gi|61652782| were identified with the highest number of recombination event (1273). However, the Mung bean yellow mosaic India virus (gi|66351988|) was found to have 1170 recombination event. The phylogenic analysis among the highly recombinant sequences was carried to get an insight of the evolution among viral sequences in this class of plant viruses. The phylogenetic analysis revealed a pattern in diversity among these virus strains and a split tree analysis showed diversity in the range of 0.049128335 to 10.269852. This in silico analysis may pave way for a greater understanding of recombination phenomenon in Ggeminiviruses and it might be helpful for strategic plant viral disease management.

Transient expression of siRNA targeted against the TYLCV AV1, AC1 and AC3 genes for high resistance in tomato

Tomato yellow leaf curl virus (TYLCV) is a globally devastating disease that affects the production of many crops, particularly tomato, leading to considerable economic losses. RNA interference, which allows sequence-specific gene silencing at the post-transcriptional level, is an effective method to obtain virus-resistant lines of crops. Agrobacterium-mediated transient expression assay has emerged as a rapid and useful approach to evaluate gene functions in plants without the need to produce transgenic lines. In this study, we transformed the plant expression vector pBIN438-AV1–AC1–AC3(i/r), which consists of the inverted repeat of ΔAV1, ΔAC1, and ΔAC3 fusion fragments into Agrobacterium tumefaciens EHA105 to generate tomato lines with high viral resistance. The viral resistance of the fusion gene was evaluated by transient expression. The test plants did not show disease symptoms at 35 d postinoculation with TYLCV. This study provided an important method for plant antiviral breeding and safe tomato production.

Characterization of Tomato yellow leaf curl virus and associated alphasatellite infecting Cucurbita maxima in Japan

Samples of Cucurbita maxima plants with foliar yellowing and curling symptoms were collected in Komae City (Japan) in 2011 and shown by Southern blot hybridization to be associated with a begomovirus. Rolling circle amplification and PCR-mediated amplification with specific primers showed the presence of a begomovirus and an alphasatellite, but neither a DNA-B component or a betasatellite. Sequence analysis of full-length clones showed the virus to be an isolate of the Israel strain of Tomato yellow leaf curl virus and the alphasatellite Sida yellow vein China alphasatellite. This is the first identification of these components in a cucurbit in Japan.

Tomato mottle wrinkle virus, a recombinant begomovirus infecting tomato in Argentina.

Begomoviruses seriously threaten tomato production in South America. Here, we present the molecular characterization of a novel tomato-infecting begomovirus isolated in Argentina and demonstrate its infectivity. After cloning and sequencing the complete genome of this new virus, pairwise genetic distance and phylogenetic analyses revealed that it is a novel virus that is closely related to other begomoviruses found in Argentina, Brazil and Bolivia. We have proposed naming the virus tomato mottle wrinkle virus (ToMoWrV), based on symptoms produced upon its biolistic inoculation into tomato plants. Recombination analysis revealed that ToMoWrV is a recombinant, with parental sequences likely belonging to the South American begomoviruses soybean blistering mosaic virus (SoBlMV) and tomato yellow vein streak virus (ToYVSV).

Temporal and spatial spread of potyvirus infection and its relationship to aphid populations visiting garlic crops

The potyviruses Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV) are the main causes of serious losses in garlic crops worldwide. Both viruses are transmitted by aphid vectors in a non-persistent manner. The relationships of aphid populations with temporal and spatial patterns of OYDV and LYSV were studied in a commercial main garlic production area from Mendoza, Argentina. The virus incidence in garlic plots during 2 years was quantified by a nitrocellulose-enzyme-linked immunosorbent assay. For temporal analyses performed in 2007 and 2008, disease progress curves were fitted using a logistic model. Epidemics were driven by non-colonising aphid species that spread the viruses primarily from west to east, coinciding with the wind pattern. This directional trend was reflected in the spatial analysis as a left-to-right gradient of virus incidence and cumulative aphid counts. Between 46 and 60 % of plants were infected with OYDV and LYSV in the first crop cycle exposed to natural infection. A checklist of aphid species visiting the garlic crop was generated, with 34 species detected. We found that total aphid catch is a better predictor of virus spread than catches of any single species or a combination of a few key species.

Potential population growth of "Melanaphis sacchari" (Zethner) reared on two hosts plants

The sugarcane aphid [ Melanaphis sacchari (Zethner) (Hemiptera: Aphididae)] feeds on several graminaceous host-plants and it is a serious pests for sugarcane ( Saccharum officinarum ) and sorghum ( Sorghum spp ). This aphid causes direct damage by suction of the plant’s sap and indirectly transmits ScYLV (Sugarcane Yellow Leaf Virus), which causes losses in ethanol and sugar production. The aim of this work was to compare biological parameters, and population growth of sugarcane aphid in its major host-plants: sweet sorghum (cv. BRS506) and sugarcane (cv. RB867515). Newborn nymphs were reared on excised leaves in climatic chambers with controlled environmental conditions. Survivorship offspring was recorded in order to obtain biological parameters derived from life tables. Some parameters were significantly different by contrast and clearly showed that sweet sorghum cv. RB867515 is more suitable for population growth of sugarcane aphid than sugarcane RB867515.

Verification of serological relationship between two phylogenetically related peanut-infecting Tospovirus species

Based on the serological relationships of nucleocapsid proteins (NPs), a tospovirus species can be classified as a member of a serogroup or a distinct serotype, which greatly helps virus identification and disease diagnosis. Recent studies reported that distinct tospovirus species sharing above 51.8 % amino acid (aa) identity in their NPs may be serologically related. Two phylogenetically related peanut-infecting tospovirus species, Peanut chlorotic fan-spot virus (PCFV) in Taiwan and Peanut yellow spot virus (PYSV) in India, were previously considered as distinct serotypes, since no serological relationship has been established. To verify the serological relationship of PCFV and PYSV, the NP of PCFV was purified from leaf tissues of the infected Chenopodium quinoa plants and used to produce polyclonal antiserum (RAs-PCFV NP) and a monoclonal antibody (MAb-PCFV NP). Polyclonal antiserum to the bacterially expressed NP of PYSV (RAs-PYSV NP) was also prepared. RAs-PCFV NP reacted with the homologous PCFV NP and the bacterial-expressed PYSV NP and tissue extracts of PYSV-infected plants. Reciprocally, RAs-PYSV NP reacted with the homologous bacterial-expressed PYSV NP and the tissue extracts of PYSV-infected or PCFV-infected plants. In addition, MAb-PCFV NP reacted only with the tissue extracts of PCFV-infected plants. Our results demonstrate that PCFV NP is serologically related to PYSV NP and they should be classified as members of a unique serogroup.

VIRUS FITOPATÓGENOS EN INSECTOS ASOCIADOS AL AJO

Vegetative propagation of garlic (Allium sativum) is the main route of virus transmission for this crop. However, insectvector spread should not be ruled out. The aim of this study was to detect, by means of the ELISA test, the presence of five viruses in insects collected in garlic plants. The experiment was conducted during the fall-winter (2008-2009) cycle. Insect samples were taken on three dates: 45, 110 and 140 days after garlic sowing. Insect species identification was performed using a Zeiss (30X) stereomicroscope and the O’Brien and Wilson (1985) and Mound and Kibby (1998) taxonomy keys. The serology test for virus detection was the DAS-ELISA technique. Coat-protein virus antibodies were applied for the potyviruses: Leek yellow spot virus (LYSV) and Onion yellow dwarf virus (OYDV); for the carlaviruses: Garlic common latent virus (GCLV) and Shallot latent virus (SLV); and for the tospovirus: Iris yellow spot virus (IYSV). Of the 19 insect species identified, Thrips tabaci Lindeman tested positive in 18 samples for GCLV and in 2 samples for IYSV, and Collops quadrimaculatus tested positive in one sample for GCLV.

PREPARATION OF ANTISERUM TO RECOMBINANT COAT PROTEIN FOR DETECTING STRAWBERRY MILD YELLOW EDGE VIRUS

Strawberry mild yellow edge virus (SMYEV) is one of the most economically important viral pathogens infecting strawberries worldwide. Immunodiagnostics is a rapid and sensitive method for detecting viruses. Preparation of antiserum against recombinant coat protein (CP) has been used for some viruses. In this paper, the full length CP gene of SMYEV, which consisted of 729 bp and encoded 242 amino acid residues, was isolated from the SMYEV isolate SY05, and then cloned into the prokaryotic expression vector pGEX-6P-1. The recombinant prokaryotic expression vector carrying the SMYEV CP gene was introduced into Escherichia coli and the fused CP protein was expressed in E. coli cells treated with IPTG. The antiserum was produced after the rabbit was immunized with the purified CP protein. Two methods, ELISA and RT-PCR, were compared for detecting SMYEV, and the results show that ELISA was as not sensitive as RT-PCR.

FIRST REPORT OF SOLANUM ELAEAGNIFOLIUM AS NATURAL HOST OF TOMATO YELLOW LEAF CURL VIRUS SPECIES (TYLCV AND TYLCSV) IN TUNISIA

Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae), as well as recombinant variants, were recently found in tomato in Tunisia (Mnari-Hattab et al., 2014). To identify sources of infection, tomato plants and Solanum elaeagnifolium, an invasive weed, were sampled and tested for the presence of TYLCV and TYLCSV. Genomic DNA was purified from leaf samples showing curling and yellowing and subjected to PCR using primers designed in the coat protein gene of TYLCV (Mnari-Hattab et al., 2014; Wyatt and Brown, 1996). A product of ca. 580 bp was obtained from four of six S. elaeagnifolium and six of six tomato samples tested. The nucleotide sequence of one amplicon from tomato and two amplicons from S. elaeagnifolium was obtained and deposited in GenBank as accession numbers KF990598, KF990599 and KF990600. The nucleotide sequence of Tunisian TYLCV isolates was 91.2 to 98.9% identical and shared 91 to 98.3% identity with a TYLCV isolate from Turkey (AJ867487). Multiplex PCR (Davino et al., 2008) was further used to identify virus species with the amplification of a 570 bp fragment for TYLCV and a 800 bp fragment for TYLCSV. Results showed two S. elaeagnifolium and four tomato plants infected with TYLCV or TYLCSV, and two S. elaeagnifolium and one tomato plant infected with both viruses. To the best of our knowledge, this is the first report of S. elaeagnifolium as a natural host of TYLCV and/or TYLCSV in Tunisia. These findings suggest S. elaeagnifolium as a reservoir for tomato yellow leaf curl virus disease.

BIOLOGICAL AND MOLECULAR STUDIES ON THE TOXIC EFFECT OF VEGETATIVE INSECTICIDAL PROTEIN (VIPs) OF Bacillus thuringiensis EGYPTIAN ISOLATES AGAINST WHITEFLIES

Whitefly Bemisia tabaci (Gennadius) transmitted geminiviruses cause epidemics in vegetable and fiber crops. It can infect more than 600 species of host plants. It also considered as the major vector transmitting various types of geminiviruses such as tomato yellow leaf curl virus (TYLCV) which cause great damage to tomatoes crop. Previous studies have reported that the vegetative insecticidal proteins (VIPs) of Bacillus thuringiensis (Bt) revealed insecticidal activity against different insect species. In this study, two local isolates of B. thuringiensis named BtC-18 and BtDI-29 were screened for the insecticidal activity of VIPs against whitefly population. Analysis of median lethal concentration (LC50) revealed that B. thuringiensis strain BtC-18 is more potent and toxic than BtDI-29 strain against whiteflies with an estimated LC50 of 90 ppm and 160 ppm for BtC-18 and BtDI-29, respectively. However, the median lethal time (LT50) value did not show significant difference between both isolates. PCR analysis of vip genes confirmed the presence of vip1, vip2 and vip3 genes on BtC-18 genome. Proteins extract from the BtC-18 culture pellet were further purified by 80% saturation of ammonium sulfate precipitation. The purified protein showed a clear band at 88 KDa corresponding to Vip3A protein as previously demonstrated. The LC50 of the purified band at 88KDa showed insecticidal activity against white fly with an estimated LC50 of 898 ppm.

토마토덤불위축바이러스 현장진단용 급속 면역 금 나노입자 막대 키트 개발

A rapid, user-friendly and simple immune-chromatographic dipstick kit named ‘rapid immuno-gold strip’ (RIGS) kit was developed in a novel single strip format to detect on-site detection of Tomato bushy stunt virus (TBSV). Immunoglobulin G (IgG) from polyclonal antisera raised in rabbits against TBSV was purified through protein-A affinity chromatography and then the purified TBSV-IgG was conjugated to colloidal gold nano-particles which served as a test line on nitrocellulose membrane. Protein A that non-specifically binds to TBSV antibody was used as a control line on the same strip. The diagnosis process with the RIGS-TBSV involves simply grinding the suspect plant sample in a bag that contains the extraction buffer and inserting the strip the bag. Results can be seen in 5-10 minutes. The flow of the complexes of gold particles coated with TBSV-IgG and a crude sap from TBSV infected tomato plants resulted in intensive color formed on the test lines proportional to the concentrations of TBSV. The RIGS-TBSV kit did not show any cross-reactions against Tomato spotted wilt virus and Tomato yellow leaf curl virus that are major causal viruses. These results indicate that the RIGS-TBSV kit is highly sensitive and is not required for laboratory training and experience prior to testing. The RIGS-TBSV kit is suitable for on-site determination of suspect TBSV-infected plants as well as for laboratory diagnosis.