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Subject: Veterinary Parasitology × Author: SANDEEP. N × End date: 2022 × Start date: 2022 × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    DEVELOPMENT OF LOOP MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) ASSAY FOR THE DIAGNOSIS OF THEILERIOSIS IN CATTLE
    (KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR - 585 401, 2022) SANDEEP. N; DHANALAKSHMI, H.)
    A study on 200 blood samples collected from different parts of Karnataka was undertaken to develop a paper based LAMP assay and a foldable isothermal amplification micro-device for the diagnosis of theileriosis in cattle. Both HNB wet LAMP and phenol red wet LAMP assays were standardized for diagnosis of Theileria annulata in cattle. Half the volume of optimized HNB wet LAMP was dried on paper substrate for the standardization of paper HNB LAMP with modification in dye concentration and this produced a color change from violet to blue. Whereas the same volume of phenol red wet LAMP was unable to produce desired color change on paper due to inherent paper character. And same standardized HNB paper LAMP was used for the standardization of foldable isothermal amplification micro-device and this produced a color change from initial violet to colorless. We fabricated and standardized a simple, cost-effective, compact and easy to operate paper LAMP and foldable isothermal amplification microdevice which can be employed for POCT and comparative evaluation was done. To state in terms of sensitivity it was found that both wet LAMP assays and paper LAMP assays could amplify as least as 10-4ng (0.1pg) of DNA. To state in terms of specificity it was found that both wet and paper LAMP assays amplified only Theileria annulata and did not amplify other closely related haemoprotozoans. All the 200 suspected blood samples were subjected to microscopy, PCR, wet LAMP assays and paper LAMP assays. It was found that 16 samples were found to be positive for theilerosis in microscopy, 20 samples were found to be positive in PCR and 23 samples were found positive in both wet and paper LAMP assays. Hence we can say that the LAMP assays (wet and paper LAMP) was found to be more sensitive (100%) when compared to microscopy (69.5%) and PCR (86.9%). The drying of LAMP reagents on paper substrate enables us to transport LAMP kits to areas where the cold chain is not easily available and employed for POCT because its single step operation.