Development of a triple antibody sandwich enzyme-linked immunosorbent assay for cassava mosaic disease detection using a monoclonal antibody to Sri Lankan cassava mosaic virus

Abstract

Background Cassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA). Methods Monoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard. Results A TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA. Conclusions Our findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

What Can Be Learnt by a Synoptic Review of Plant Disease Epidemics and Outbreaks Published in 2021

“…Studies concerned with aspects of disease management accounted for many of the articles recovered under Objective 1, very often linked to disease surveillance in regulated zones (Lazaro et al 2021) and detection/ discrimination of new pathogens or novel strains using molecular diagnostics. Examples include Sri Lankan cassava mosaic virus emerging in Southeast Asia (Charoenvilaisiri et al 2021), Xylella subspp in the Balearic Islands (Olmo et al 2021), citrus tristeza virus strains (Park et al 2021, Llanes-Alvarez et al 2021, Erwinia spp in the USA (Singh et al 2021), antibiotic resistance in Pseudomonas syringae pv actinidiae (Psa) (Lee et al, 2021), fungicide resistance in Zymoseptoria tritici (Garnault et al, 2021) (see detailed summaries in Electronic Supporting Information, S5). Many studies were concerned with the field performance and deployment of resistant varieties: such as for pokkah boeng disease in sugarcane (Shan et al 2021, Solic-Palacios et al 2021, wheat yellow rust (Perronne et al 2021, Esmail 2021.…”

A Synoptic Review of Plant Disease Epidemics and Outbreaks Published in 2021

“…About 95% of infection was estimated as due to whitefly transmission rather than through infected stem cuttings, the original source of infection. A TAS-ELISA for SLCMV detection in southeast Asia has been developed in Thailand and was shown to have 99.6% accuracy and 100% when tested with stem cuttings derived from 490 field samples collected between 2018 and 2019 (Charoenvilaisiri et al 2021). The status of cassava mosaic disease caused by SLCMV in Southeast Asia has been reviewed (Om, et al 2021).…”