BBrMV is a serious constraint in the production of banana and plantain in India [14] and the Philippines [10]

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 production 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. Effective management of BBrMV needs rapid and reliable diagnostic methods to detect virus infection. Currently, most banana virus researchers have focused on laboratory dependent diagnostic techniques such as ELISA, RT-PCR, real-time PCR and RT-LAMP, which are very useful in larger laboratories with appropriate equipments but are not suitable for onsite or field use because of the requirement for skilled technicians. Hence, there is a need to detect the disease in a rapid, simple and field-usable manner and effectively to manage an outbreak of a viral disease in the field, which was the primary focus of our current investigation. In this study, we developed a LFIA for the rapid detection of BBrMV.
We expressed and purified BBrMV antigen (CP) in E. coli and used recombinant CP to immunize the rabbit. Compared to virus particle purification in banana which has high poly phenols and poly saccharides, the strategy of prokaryotic expression provides a fast approach to generate a high yield of BBrMV antigens. The sensitivity and specificity of LFIA crucially depend on the antibodies used in immunostrip. We used affinity purified antibody in test line as well as for the conjugation with 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 crucial 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 prepared 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 developed LFIA 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 and validation of the assay in the field proved that the specificity and sensitivity of the developed LFIA were comparable to ELISA, for the samples tested. The Cohen’s kappa coefficient (0.94) indicates very good concordance between the LFIA developed in the present study and ELISA. A high negative predictive value indicates that the chances of getting a false negative result are remote in case of the developed LFIA. Moreover, Cohen’s kappa test was conducted to calculate the degree of agreement between the results obtained using the above two assays 6. Therefore, the performance of this developed assay was comparable with that of the ELISA kit in analyzing leaf samples. Although ELISA is a sensitive and reliable method, it requires expensive equipment, trained personnel and a wet lab to perform it. In contrast, this developed LFIA involves no trained technians and is user-friendly; therefore, it is better suited for routine on-site (or) in field testing of BBrMV. NCS-TCP is a dynamic and comprehensive system intended for facilitating production of quality tissue culture plants and providing mechanisms for certification of quality tissue culture plants in India. Till date, a total of 283 million TC banana plants were certified from the DBT accredited test laboratory of ICAR-NRCB, (R. Selvarajan unpublished). Since 2007, both the mother plant and TC raised banana plant are being tested against four banana infecting viruses as part of certification for quality planting materials under NCS-TCP in India. Now, this LFIA strips developed in this study is recommended for indexing in place of ELISA for detection of BBrMV.
In conclusion, LFIA can be completed in field conditions within 5-10 min without any help of skilled workers and special equipments and the results are easily interpreted. It can be used for on-site diagnosis in banana plantations and nurseries especially for TC companies who collect mother plants of banana for mass propagation. This characteristic promotes self-diagnosis and elimination of infected materials by the banana growers, tissue culture industries personnel’s and nursery workers. Furthermore, the BBrMV-LFIA kit can be used to educate banana growers and stakeholders on viral diseases. The BBrMV-LFIA kit has a shelf life of at least one year (data not shown). This characteristic allows the kit to be transported easily over long-distance and to be used all over the world. The LFIA was highly specific and sensitive and was able to detect BBrMV at low concentrations. This assay provides a simple, fast and effective method for the detection of BBrMV and can be applied to the diagnosis and surveillance of viruses in the field. To the best of our knowledge, this is the first report on onsite LFIA test for detection of BBrMV in both field and laboratory samples.