BBrMV diagnostic methods to detect virus infection for

BBrMV is a serious constraint in the production of banana and plantain in India 14 and the Philippines 10. BBrMV poses a considerable quarantine risk due to its limited geographic distribution, its ability to spread easily via the use of vegetative plant parts, aphid vectors, and its inconsistent expression of visible symptoms 10,17. In the absence of any control measures, prevention of infection is the only option available to check the spread of the disease Kulabhusan et al., 2017. Effective management of banana viruses need rapid and reliable diagnostic methods to detect virus infection for timely implementation of control measures. Currently, most of the plant virologists have focused on laboratory dependent diagnostic techniques either ELISA or PCR, which are very useful in larger laboratories with appropriate equipments but are not adoptable for the on-site or in-field use because of the requirement for highly skilled technicians. Therefore, the present study focused on developing a rapid, simple and field-usable kit for BBrMV to effectively to manage an outbreak of this naturally important viral disease in the field.
In this investigation, we expressed and purified BBrMV CP in E. coli and used to immunize the rabbit as the virus purification in banana is cumbersome as it has high quantity of poly phenols and poly saccharides. The strategy of prokaryotic expression of BBrMV antigens provides a fast approach to generate a high yield of sensitivity and specificity. In LFIA, quality of antibodies or the IgG play a crucial role in achieving the sensitivity and specificity. We used affinity purified IgG for printing in test line as well as for the conjugating with the colloidal gold. Preparation of high quality colloidal gold solution is a key step to ensure the sensitivity of the LFIA strip . Furthermore, optimal pH and particle size of the colloidal gold and concentration of antibody are also critical for the development of an effective LFIA test, as they would affect the performance and reliability of the colloidal gold-antibody conjugate 19. Our finding indicates that the colloidal gold particles with a mean diameter of 30 nm and an optimum pH of 9.0 and they combined stably with antibody at 16 ?g/ml concentration. LFIA technique has been widely applied to detect other plant viruses 3-5, 8-9,11-12,18-19,21. The LFIA developed in this study, detected the virus at 1:20 dilution of BBrMV infected sap. This method can detect BBrMV not only in plant leaf tissues but also in different floral parts and seeds (data not shown).
Our investigation on the limit of its detection and specificity are comparable to that of ELISA as evidenced from the validation of the strips to detect large number of field samples. Cohen’s kappa test was conducted to calculate the level of agreement between the results obtained using the ELISA and LFIA 6. Further, the Cohen’s kappa coefficient of 0.94 indicates a higher confidence on these two assays prepared. There is a very remote possibility of obtaining false negative, as the negative prediction value is high (1.0). Though ELISA is a method it requires equipments, laboratory environment, trained personnel and a reliable relatively easy to perform and sensitive compared to LFIA which involves no trained technical personnel and is user-friendly; therefore, it is a highly suitable method for routine on-site (or) in field testing of BBrMV.

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