RESUMO
OBJECTIVE@#To establish a suitable method of diagnosis of visceral leishmaniasis (VL) using peripheral blood, spleen or bone marrow aspirates.@*METHODS@#Peripheral blood, bone marrow and spleen aspirate samples were collected from clinically suspected VL patients (n = 26). A new PCR primer pair (MK1F/R) was designed targeting kinetoplast mini circle DNA sequences of Leishmania donovani, and Leishmania infantum, and was used to diagnose VL along with some other established primers for VL in polymerase chain reactions. Test was validated by comparing with several other diagnostic methods.@*RESULTS@#The designed primer set showed 100% specificity and 98% sensitivity in detecting VL using blood samples, when compared with more invasive samples: bone marrow or spleen aspirates.@*CONCLUSIONS@#The newly designed primer MK1F/R could be a better alternative for PCR based diagnosis of VL using less invasive sample, peripheral blood instead of bone marrow or spleen aspirates.
RESUMO
Objective To establish a suitable method of diagnosis of visceral leishmaniasis (VL) using peripheral blood, spleen or bone marrow aspirates. Methods Peripheral blood, bone marrow and spleen aspirate samples were collected from clinically suspected VL patients (n = 26). A new PCR primer pair (MK1F/R) was designed targeting kinetoplast mini circle DNA sequences of Leishmania donovani, and Leishmania infantum, and was used to diagnose VL along with some other established primers for VL in polymerase chain reactions. Test was validated by comparing with several other diagnostic methods. Results The designed primer set showed 100% specificity and 98% sensitivity in detecting VL using blood samples, when compared with more invasive samples: bone marrow or spleen aspirates. Conclusions The newly designed primer MK1F/R could be a better alternative for PCR based diagnosis of VL using less invasive sample, peripheral blood instead of bone marrow or spleen aspirates.
RESUMO
An intracellular protease was extracted and purified from Pseudomonas aeruginosa by ion-exchange chromatography on DEAE-cellulose followed by CM"cellulose and rechromatography on DEAE-cellulose. The purified protease was found to be homogeneous as judged by polyacrylamide disc gel electrophoresis [PAGE]. The molecular mass of the protease as determined by gel filtration on G-150 was about 48,000 and about 49,000 on SDS-PAGE. The enzyme is monomeric in nature. The purified protease is a glycoprotein with neutral sugar content of 0.6%. The Km value of the protease was found to be 0.48% against casein as substrate. The enzyme is stable up to 600C and showed maximum activity around 500C. The enzyme activity was affected with the changes of pH and the maximum proteolytic activity was observed at pH 8.0. The protease activity was inhibited in the presence of EDTA, Cu2+, Mn2+and Hg2+ whereas the presence of Ca2+, K+, Na+ and ascorbic acid enhanced the activity