Detection of tomato spotted wilt virus in chrysanthemum by immunocapture RT-PCR assay.

Fuji, Shin-ichi; Ohishi, Kazushi; Nakamae, H.

doi:10.4165/kapps1958.40.0_111

Cited by 3 publications

(4 citation statements)

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“…Several diagnostic methods have been developed for the detection of TSWV: enzyme‐linked immunosorbent assay (ELISA) using polyclonal antibodies (PAbs) (Cho et al., 1988; Ghotbi et al., 2005) and monoclonal antibodies (MAbs) (Sherwood et al., 1989; Huguenot et al., 1990), dot‐immunobinding assay (Wang and Gonsalves, 1990), reverse transcription‐polymerase chain reaction (RT‐PCR) (Dewey et al., 1996; Okuda and Hanada, 2001), immunocapture RT‐PCR (IC‐RT‐PCR) (Fuji et al., 1998; Fukuta et al., 2004) and real time RT‐PCR (Roberts et al., 2000). Of these, ELISA, RT‐PCR and IC‐RT‐PCR are most widely used.…”

Section: Introductionmentioning

confidence: 99%

Monoclonal Antibodies against the Recombinant Nucleocapsid Protein of Tomato spotted wilt virus and its Application in Virus Detection

Wu¹,

Yang²,

Zhou³

2009

Journal of Phytopathology

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Tomato spotted wilt virus (TSWV) is the type member of the tospovirus genus and causes significant losses in a wide range of economically important ornamental and vegetable crops worldwide. The nucleocapsid gene, located on the ambisense S RNA segment of TSWV was expressed in Escherichia coli using pET-32a as vector and correct expression of recombinant protein was confirmed by Western blot using an anti-TSWV monoclonal antibody (MAb). The recombinant protein was purified using Ni-NTA agarose and the purified protein was used for the production of MAbs. Three murine MAbs against the recombinant nucleocapsid protein were produced. Triple antibody sandwich enzyme-linked immunosorbent assay and immunocapture RT-PCR methods were then established for reliable and efficient detection of TSWV using the produced MAbs.

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Section: Introductionmentioning

confidence: 99%

Monoclonal Antibodies against the Recombinant Nucleocapsid Protein of Tomato spotted wilt virus and its Application in Virus Detection

Wu¹,

Yang²,

Zhou³

2009

Journal of Phytopathology

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“…It has been reported that the detection sensitivities of TSWV in chrysanthemum using RNA extraction RT‐PCR (Matsuura et al., 2002) and dot‐blot hybridization of PCR products (Fuji et al., 1997, 1998) were higher than that of ELISA. We therefore used RNA extraction RT‐PCR combined with dot‐blot hybridization to determine the presence of low concentrations of virus in cuttings.…”

Section: Discussionmentioning

confidence: 99%

“…The remaining water extracts were subjected to DAS‐ELISA in the same way as described above. RT‐PCR was performed as described previously (Matsuura et al., 2002) using two opposing primers to amplify 457 bp fragments of NSm protein gene designed by Fuji et al. (1998) on the basis of the nucleotide sequence of RNA segment M (Kormelink et al., 1992) of TSWV.…”

Section: Methodsmentioning

confidence: 99%

Localization of Tomato spotted wilt virus in Chrysanthemum Stock Plants and Efficiency of Viral Transmission from Infected Stock Plants to Cuttings

Matsuura¹,

Ishikura²,

Shigemoto³

et al. 2004

Journal of Phytopathology

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Localization of Tomato spotted wilt virus (TSWV) in susceptible chrysanthemum stock plants and its efficiency of transmission from latently infected stock plants to cuttings under thrips-free conditions were investigated using double-antibody sandwich enzymelinked immunosorbent assay (DAS-ELISA), reverse transcription polymerase chain reaction (RT-PCR) combined with dot-blot hybridization, and by cultivation. TSWV localized in symptomless stock plants and cuttings taken from them, and its detection frequency, was relatively high in the lower leaves and stems, but low in terminal buds of cuttings. In cv. Seinoyakata, the detection rate of TSWV in cuttings taken from infected stock plants ranged from 26.3 to 38.5% in 2002 and from 16.7 to 39.1% in 2003, and in cv. Jimba from 37.5 to 47.1% from January to May 2003. No fluctuation in detection rate by cutting generation or season was observed. The detection rate of TSWV in the roots of stock plants was >50% in most periods. These results indicate that transmission efficiency from infected chrysanthemum stock plants to cuttings is fairly high (approximately 20-50%) and thus may become a major source of secondary infection in production fields or glasshouses. Our results also suggest that several lower leaves should be combined in ELISA to detect infection in stock plants and cuttings.

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“…TSWV causes economic losses to a wide of economically important crops and particularly to vegetables such as peppers and tomatoes in Korea (Cho et al, 2005;Choi et al, 2010;Kim et al, 2004). Various immune-assay methods have been reported for detection of TSWV, such as enzyme-linked immunosorbent assay (Adam et al, 1996;de Avila et al, 1990;Gonsalves and Trujillo, 1986;Huguenot et al, 1990;Sherwood et al, 1989), dotimmunobinding assay (Wang and Gonsalves, 1990), immunecapture Reverse-transcription (RT)-PCR (Fuji et al, 1998;Fukuta 16 et al, 2004;Weekes et al, 1996), RT-PCR (Dewey et al, 1996;Mumford et al, 1996;Okuda and Hanada, 2001;Sharma et al, 2000), and real-time RT-PCR (Boonham et al, 2002;Debreczeni et al, 2011;Robert et al, 2000). However, this kind of determination required some expensive equipment, for example, a microplate reader.…”

Section: Introductionmentioning

confidence: 99%

Development of Rapid Immune-gold Strip Kit for On-Site Diagnosis of Tomato spotted wilt virus

Yoon¹,

Choi²,

Cho³

et al. 2014

Research in Plant Disease

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A rapid, user-friendly and simple immune-chromatographic dipstick kit named 'rapid immune-gold strip' (RIGS) kit was developed in a novel single strip format to detect on-site detection of Tomato spotted wilt virus (TSWV). Immunoglobulin G (IgG) from polyclonal antisera raised in rabbits against TSWV was purified through protein-A affinity chromatography and then the purified TSWV-IgG was conjugated to colloidal gold nano-particles which served as a test line on nitrocellulose membrane. Protein A that non-specifically binds to TSWV antibody was used as a control line on the same strip. The diagnosis process with the TSWV-RIGS 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 2-5 minutes. The flow of the complexes of gold particles coated with TSWV-IgG and a crude sap from TSWV-infected pepper, tobacco and tomato plants resulted in intensive color formed on the test lines proportional to the concentrations of TSWV. The RIGS-TSWV kit did not show any cross-reactions against other tomato-infecting viruses unrelated to TSWV. These results indicate that the TSWV-RIGS kit is highly sensitive and is not required for laboratory training and experience prior to testing. The TSWV-RIGS kit is suitable for on-site detection of suspect TSWV-infected plants as well as for laboratory diagnosis.

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Detection of tomato spotted wilt virus in chrysanthemum by immunocapture RT-PCR assay.

Cited by 3 publications

References 0 publications

Monoclonal Antibodies against the Recombinant Nucleocapsid Protein of Tomato spotted wilt virus and its Application in Virus Detection

Monoclonal Antibodies against the Recombinant Nucleocapsid Protein of Tomato spotted wilt virus and its Application in Virus Detection

Localization of Tomato spotted wilt virus in Chrysanthemum Stock Plants and Efficiency of Viral Transmission from Infected Stock Plants to Cuttings

Development of Rapid Immune-gold Strip Kit for On-Site Diagnosis of Tomato spotted wilt virus

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