A multiplex RT-PCR-ELISA to identify plant pathogens

Tospovirus infects and causes severe damage to a wide variety of plants worldwide. In Thailand, four tospovirus species have been reported in the field, namely Capsicum chlorosis virus (CaCV), Melon yellow spot virus (MYSV), Tomato necrotic ringspot virus (TNRV) and Watermelon silver mottle virus (WSMoV). For the purposes of molecular biology and epidemiology studies, diagnosis, disease management, and the selection of tospovirus-resistant plants in breeding programs, accurate tospovirus detection and identification systems are very important.
 
BIOTEC research team developed a multiplex RT-PCR-ELISA to detect and differentiate four tospovirus species, namely CaCV, MYSV, TNRV), and WSMoV. A universal primer pair targeting the well-characterized nucleocapsid (N) gene was designed and used for simultaneous amplification and DIG-labeling of four tospovirus N gene sequences by RT-PCR. The DIG-labeled N-gene PCR products were then differentiated into species by four parallel microwell-captured specific probe hybridizations and detected by ELISA.
Multiplex PCR
The developed assay has superior specificity, sensitivity, and high-throughput capacity compared to conventional RT-PCR. With appropriate primer and probe design, the capacity of the multiplex RT-PCR-ELISA developed in this study can easily be extended to the identification of other tospovirus species.
 
This work is a collaboration between researchers from Monoclonal Antibody Production Laboratory , Plant Research Laboratory and Microbial Cell Factory Laboratory.
 
Reference:
Charoenvilaisiri, S., Seepiban, C., Bhunchoth, A., Warin, N., Luxananil, P. and Gajanandana, O.  Development of a multiplex RT-PCR-ELISA to identify four distinct species of tospovirus. Journal of Virological Methods 202, 54-63 (2014)
 
Posted on 2 July 2014

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