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Optimization Of Loop-Mediated Isothermal Amplification (Lamp) For The Molecular Diagnosis Of Feline Babesiosis

By: Muhammad Awais Salim (2012-va-606) | Dr. Muhammad Lateef.
Contributor(s): Prof. Dr. Azhar Maqbool | Dr. Muhammad Nauman Zahid.
Material type: materialTypeLabelBookPublisher: 2015Description: 27p.Subject(s): Department of ParasitologhyDDC classification: 2374-T Dissertation note: Babesia is a worldwide tick borne hemoparasite causing Babesiosis, an important disease affecting a number of animals and attracting the researcher’s attention due to its zoonotic potential.Babesiosis in cats often presents as a chronic and low grade disease, however most common symptoms include anaemia, lethargy, weakness and rarely icterus and fever. Blood samples were collected from 100 domestic cats at Pet Center, UVAS, Lahore,from their ear tips and cephalic/saphenous vein. The blood will be immediately transferred to EDTA coated vacutainers. Stained thin blood smears were observed for intra-erythrocytic bodies and 45 samples were selected after screening. Blood in EDTA were tested for PCR (already optimized) in the Molecular Parasitology laboratory at UVAS, Lahore, to screen for B. felis. ExtractedDNA of confirmed B. felis samples were further processed forLoop-Mediated Isothermal Amplification (LAMP). LAMP primers weredesigned recognizing four sections of the B. felis gene. LAMP reactions of 25µL were standardized at 60°C temperature for 1 hour time using DNA extracted from blood samples of cats found positive on PCR. Briefly, the concentration of FIP and BIP were varied from 0.8µM to 2.4µM, Mg2+from 2mM to 4mM, betaine from 0.2 to 0.8M and dNTPs from 1mM to 4mM.The LAMP reaction was optimized at the final concentration of 0.2µM F3 and B3, 2.0µM of each of the FIP & BIP, 2mM for each dNTPs, 0.8M betaine, 1X reaction buffer, 1µl bst polymerase and 2µl DNA templates at 60°C.
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Babesia is a worldwide tick borne hemoparasite causing Babesiosis, an important disease affecting a number of animals and attracting the researcher’s attention due to its zoonotic potential.Babesiosis in cats often presents as a chronic and low grade disease, however most common symptoms include anaemia, lethargy, weakness and rarely icterus and fever.
Blood samples were collected from 100 domestic cats at Pet Center, UVAS, Lahore,from their ear tips and cephalic/saphenous vein. The blood will be immediately transferred to EDTA coated vacutainers. Stained thin blood smears were observed for intra-erythrocytic bodies and 45 samples were selected after screening. Blood in EDTA were tested for PCR (already optimized) in the Molecular Parasitology laboratory at UVAS, Lahore, to screen for B. felis. ExtractedDNA of confirmed B. felis samples were further processed forLoop-Mediated Isothermal Amplification (LAMP).
LAMP primers weredesigned recognizing four sections of the B. felis gene. LAMP reactions of 25µL were standardized at 60°C temperature for 1 hour time using DNA extracted from blood samples of cats found positive on PCR.
Briefly, the concentration of FIP and BIP were varied from 0.8µM to 2.4µM, Mg2+from 2mM to 4mM, betaine from 0.2 to 0.8M and dNTPs from 1mM to 4mM.The LAMP reaction was optimized at the final concentration of 0.2µM F3 and B3, 2.0µM of each of the FIP & BIP, 2mM for each dNTPs, 0.8M betaine, 1X reaction buffer, 1µl bst polymerase and 2µl DNA templates at 60°C.

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