Abstract
BackgroundCassava 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).MethodsMonoclonal 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.ResultsA 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.ConclusionsOur 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.
Highlights
Cassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia
Our findings suggest that the TAS-ELISA for Sri Lankan cassava mosaic virus (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
Polymerase chain reaction (PCR) using previously published SLCMV-specific primers was found to detect SLCMV in infected cassava extracts diluted at 1:5,242,880. These results indicated that the newly developed TAS-ELISA based on polyclonal antibody (PAb) PK and Monoclonal antibodies (MAbs) 29B3 can be considered a highly sensitive method for SLCMV detection in infected cassava leaf samples, with a detection limit of 256 times greater than that of a commercial ELISA kit, and 64 times less than that of PCR
Summary
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). Besides serving as a major staple food in developing countries, it serves as the raw materials for various starch-based industries in many regions of the world [2]. Cassava mosaic disease (CMD) caused by a complex of cassava mosaic begomoviruses (CMBs; genus Begomovirus, family Geminiviridae) represents one of the major constraints to cassava production in many cassava-growing countries in Africa and Asia [4]. Eleven CMB species have been identified, nine in Africa and two in Asia [6]
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