International Postgraduate Research Conference (IPRC)
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Item Assessment of Real Time Polymerase Chain Reaction (PCR) Assay for the Early Diagnosis of Leptospirosis in Humans(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Uduwawala, U.M.H.U.; Manamperi, A.; Gunaratna, G.P.S.; Karunanayake, L.; Chandani, W.L.; Hapugoda, M.Leptospirosis is the most widespread zoonotic disease worldwide having a great impact on health issues in developing countries. It is caused by a pathogenic spirochete of the genus Leptospira where humans become infected through contact with the urine of infected animals. It is often exceptionally under-recognized as the clinical manifestation mimics variety of similar disease conditions that occur in the same environmental and climatologic conditions which accentuate the importance of laboratory diagnosis of leptospirosis. At present, no hospital based facilities are available for acute confirmation of the disease. The existing practice is retrospective confirmation with serological diagnosis. Therefore, the establishment of acute phase diagnosis will help in monitoring the disease, determining when hospital admission is required and reduce case fatalities. The objective of this study was to establish and evaluate a molecular-based assay to provide laboratory confirmation of leptospirosis at the acute phase of the infection (1-5 days of fever). Patients fulfilling clinical criteria stipulated by the accepted case definition were selected for the study and patients who failed to show evidence of sero conversion were considered as true negatives. A real time Polymerase Chain Reaction (PCR) assay with targeting a 203 bp fragment in the secY gene which is conserved among pathogenic serovars of Leptospira was established using a reference DNA sample (L.interrogans serovar Icterohaemorrhagiae strain RGA). Analytical sensitivity and the analytical specificity of the assay were calculated. The accuracy of the real time PCR was determined by a panel of acute blood samples collected from laboratory confirmed leptospirosis patients (n=35) and non-leptospirosis (n=44) patients based on Microscopic Agglutination Test (MAT) and/or IgM immunochromatography. Patients who failed to give positive test results either with MAT or IgM immunochromatography were considered as true negatives. Analytical sensitivity was approximately 314 genome equivalents per reaction and analytical specificity showed no amplification of Leptospira saprophytic sp. and other micro-organisms. The assay could effectively detect Leptospira DNA from clinically diagnosed leptospirosis suspected patients with 60.0% (21/35) diagnostic sensitivity and 77.27% (34/44) diagnostic specificity. This may be attributed to some samples failing laboratory confirmation despite their collection based on clinical suspicion. Therefore, real time PCR established can be used for rapid and definitive diagnosis of leptospirosis during the acute phase of infectionItem Use of molecular features for identification of isolated fungal pathogens of big onion damping off disease and Trichoderma spp. isolated from soil(Faculty of Graduate Studies, University of Kelaniya, 2015) Gunaratna, L.N.R.; Deshappriya, N.; Jayaratne, D.L.Big onion (Allium cepa L.) is one of the economically important spices grown in Sri Lanka. Damping off disease caused by Fusarium sp. during nursery stage of growth poses a major factor that affect the yield significantly. Application of fungicides decrease incidence of damping off disease considerably, but this is neither economical nor environmental friendly. Thus, disease management practices have to be directed towards biological control strategies. Trichoderma spp. have been extensively studied as biological control agents for controlling numerous soil-borne fungal pathogens. In the present study, isolation and identification of fungal pathogens associated with damping off disease of onion and Trichoderma spp. present in soil of the same onion fields was carried out with a view to using the Trichoderma spp. in the management of damping off pathogens. Pathogens associated with damping off were isolated from diseased and healthy seedlings (7-30 days old) collected from the fields in the Matale and Anuradhapura districts. Seedlings were surface sterilized and plated in Potato Dextrose Agar (PDA) supplemented with tetracycline. Soil samples collected from the same fields were used for the isolation of Trichoderma spp. using the Warcup method. Based on morphological characteristics and using identification keys, the fungal pathogens isolated from seedlings were identified as Fusarium, Curvularia, Alternaria and Sclerotium spp. and 14 fungal species isolated from soil samples were identified as Trichoderma spp. Although fungi can be identified using morphological features, the use of molecular biological methods tend to be more accurate. Therefore, the identity of isolated fungal species was confirmed by molecular biological methods. Genomic DNA of Fusarium spp., Alternaria spp., Trichoderma spp. were extracted. Molecular characterization of these DNA was carried out using Polymerase Chain Reaction (PCR) where the Internal Transcribed Spacer (ITS) region of rDNA gene was amplified using ITS-1 and ITS-4 primer pairs. The products were subjected to agarose gel electrophoresis. The procedures were repeated 3 times. Results showed 550 bp size bands characteristic of Fusarium spp. and 570 bp products specific to Alternaria spp. confirming the previous identity using culture based methods. Fungal species isolated from soil showed products of 600 bp which corresponds to Trichoderma sp. Molecular characterization of the potential biocontrol agents i.e. Trichoderma spp. and A.cepa L. pathogens using PCR amplification of ITS region confirmed the preliminary identities carried out using culture based methods.