title: Development of a multiplex RT-PCR-ELISA to identify four distinct species of tospovirus.
abstract: In this study, a multiplex RT-PCR-ELISA was developed to detect and differentiate four tospovirus species found in Thailand, namely Capsicum chlorosis virus (CaCV), Melon yellow spot virus (MYSV), Tomato necrotic ringspot virus (TNRV), and Watermelon silver mottle virus (WSMoV). In this system, nucleocapsid (N) gene fragments of four tospoviruses were simultaneously amplified and labeled with digoxigenin (DIG) in a single RT-PCR reaction using a pair of degenerate primers binding to the same conserved regions in all four tospovirus N genes. The DIG-labeled amplicons were distinguished into species by four parallel hybridizations to species-specific biotinylated probes in streptavidin-coated microtiter wells followed by ELISA detection using a peroxidase-conjugated anti-DIG antibody. Results indicated that the multiplex RT-PCR-ELISA assay could specifically identify each of these four tospoviruses without cross-reactivity between species or reactivity to healthy plant negative controls. Assay sensitivity was 10- to 1000-fold higher than conventional RT-PCR. When applied to naturally infected plants, all samples yielded concordant results between RT-PCR-ELISA and the reference RT-PCR. In conclusion, the multiplex RT-PCR-ELISA developed in this study has superior specificity, sensitivity, and high-throughput capacity compared to conventional RT-PCR and is an attractive alternative for the identification of different tospovirus species.
authors.affiliation: Monoclonal Antibody Production Laboratory, Agricultural Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani 12120, Thailand. Electronic address: Saengsoon.cha@biotec.or.th.
authors.forename: Saengsoon
authors.initials: S
authors.lastname: Charoenvilaisiri
authors.affiliation: Monoclonal Antibody Production Laboratory, Agricultural Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani 12120, Thailand.
authors.forename: Channarong
authors.initials: C
authors.lastname: Seepiban
authors.affiliation: Plant Research Laboratory, Agricultural Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, Kamphaengsaen, Nakornpathom, Thailand.
authors.forename: Anjana
authors.initials: A
authors.lastname: Bhunchoth
authors.affiliation: Plant Research Laboratory, Agricultural Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, Kamphaengsaen, Nakornpathom, Thailand.
authors.forename: Nuchnard
authors.initials: N
authors.lastname: Warin
authors.affiliation: Microbial Cell Factory Laboratory, Bioresource Technology Unit, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani 12120, Thailand.
authors.forename: Plearnpis
authors.initials: P
authors.lastname: Luxananil
authors.affiliation: Monoclonal Antibody Production Laboratory, Agricultural Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani 12120, Thailand.
authors.forename: Oraprapai
authors.initials: O
authors.lastname: Gajanandana
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D057166:High-Throughput Screening Assays; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009693:Nucleic Acid Hybridization; D019251:Nucleocapsid / Q000235:genetics; D010944:Plants; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013785:Thailand; D017865:Tospovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
keywords: Multiplex; Nucleocapsid gene; RT-PCR-ELISA; Tospovirus