Browsing by Author "Athapaththu, A.M.M.H."

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    DNA Fingerprinting of Thunnus obesus and Thunnus albacores Fish Species for Proper Identification in Large Scale Fish Processing Industry
    (Uva Wellassa University of Sri Lanka, 2016) Perera, D.R.C.; Gunathilaka, P.A.D.H.N.; Rodrigo, W.W.P.; Athapaththu, A.M.M.H.; Bulumulla, P.B.A.I.K.
    Detection of species substitution has become an important topic within the food industry and there is a growing need for rapid, reliable, and reproducible tests to verify species in commercial fish and seafood products. The effects of species substitution are far-reaching and include economic fraud, health hazards, and illegal trade of protected species. In Sri Lanka tuna fish industry is a rapid developing field. However, the species identification prior to the processing is achieved through morphological characteristics, which is not a reliable method. Therefore, the aim of this study was to develop a diagnostic method by combining Polymerase Chain Reaction with Restriction digestion to differentiate Thunnus obesus (bigeye tuna) and Thunnus albacores (yellowfin tuna) species in order to facilitate the fish processing industries and fish exporters by developing the test for species confirmation. Deoxy ribonucleic acid (DNA) extracted from muscle tissues of T obesus and T albacores were analyzed. DNA was amplified using primers flanking a region of cytochrome b gene of 558 by and digested using two restriction endonucleases, EcoNI and Scat A product having band sizes of 187 by and 371 by was observed from T albacores after digesting with EcoNI. The digestive product by Scal resulted 215 by and 343 by band sizes for both T albacores and T obesus. The polymorphism of DNA profiles obtained by restriction digestion was used to differentiate the T albacores and T obesus species. Therefore, the current study carries a reliable approach to identify and distinguish T obesus and T albacores from the other tuna species. Keywords: Tuna species, DNA extraction, Polymerase chain reaction, Restriction Enzyme digestion
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    Expression of a Rabies Virus Specific Antigen by Cloning the Glycoprotein Gene into Escherichia con Expression System
    (Uva Wellassa University of Sri Lanka, 2016) Sewwandi, H.S.; Rodrigo, W.W.P.; Athapaththu, A.M.M.H.; Gunathilaka, P.A.D.H.N.; Bandara, K.G.W.W.; Wijesundara, R.R.M.K.K.; Bulumulla, P.B.A.I.K.
    Rabies is an infectious disease characterized by dysfunction of the central nervous system caused by Lyssavirus of family Rhabdoviridae. Detection of rabies antibodies are used to confirm if people have been successfully immunized. Currently, these detection methods require lots of expertise and are generally carried out in reference laboratories at a high cost. Therefore, it is vital to develop and standardize simple techniques such as Enzyme Linked Immunosorbent Assay (ELISA) for determining the level of antibodies against rabies virus at a lower cost. Hence, the aim of the present study was to clone rabies virus specific glycoprotein gene into bacterial expression vector for the production of recombinant protein. Initial attempts were made to isolate plasmid DNA of pET-28a (+) vector and pcDNA3. -RVG recombinant plasmid containing previously cloned Rabies Virus Glycoprotein gene (RVG). Both plasmids were successfully digested with BamHI and XhoI restriction enzymes. The purified Rabies Virus Glycoprotein gene was cloned into pET-28a (+) bacterial expression vector. The pET-28a (+)-RVG plasmids were successfully transformed into TOPIOP competent cells through electroporation. Transformants were screened by rapid screening method. Out of 20 colonies 8 were identified as recombinants. Further screening of recombinant colonies will be carried out by digesting with restriction enzymes. Putative correct recombinant construct will be transferred into bacterial expression system for the expression. Keywords: Rabies, Rabies virus glycoprotein gene, Cloning
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    Optimization of a Polymerase Chain Reaction Based Technique to Detect Genetically Modified Foods
    (Uva Wellassa University of Sri Lanka, 2016) Thalwattal, T.G.V.N.; Rodrigo, W.W.P.; Achala, H.H.K.; Withana, W.T.G.S.L.; Athapaththu, A.M.M.H.; Bulumulla, P.B.A.I.K.
    Genetically Modified foods are an important outcome in the genetic improvement procedures in plants. Nowadays it has become a significant problem regarding authentication of such foods since non-labelled genetically modified foods are existing in the market. The aim of this study was to optimize a Polymerase Chain Reaction (PCR) based technique to detect Cauliflower Mossaic Virus 35S promoter and Nopaline Synthase terminator, which are intentionally introduced to various crops to create genetically modified foods. Three pairs of primers were used for PCR amplification. Chloroplast tRNA primers were used to amplify chloroplast DNA with 571 bp amplicon length to prove the presence of plant origin DNA. Cauliflower Mossaic Virus 35S and Nopaline Synthase forward and reverse primers with 243 and 118 by amplicon length were respectively used to detect promoter and terminator regions. PCR optimized condition for CaMV 35S promoter (annealing condition- 56 °C, 40 sec.) and NOS terminator (annealing condition- 62 °C, 30 sec.) was carried out in 30 cycles each. Fresh and processed food samples (10 each) were collected from super markets and were analyzed in triplicates. During the analysis of post PCR products using 1% agarose gel, four food samples including corn, biscuit, corn flakes and processed potato samples were detected positive for promoter and terminator regions while a processed cereal mixture was detected as positive only for the terminator region. None of the foods were labelled as GM and it indicates that non labelled genetically modified foods are presence in the market. Therefore, this method could be used as simple and reliable assay for screening of unauthorized genetically modified crops and the processed food products. Keywords: Genetically modified foods, Cauliflower mossaic virus 35S promoter, Nopaline synthase terminator, Polymerase chain reaction
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    Optimization of a Ribonucleic Acid (RNA) Extraction Protocol for Viruses in Clinical Samples for Disease Diagnosis
    (Uva Wellassa University of Sri Lanka, 2016) Kumar, S.A.; Gunathilaka, P.A.D.H.N.; Rodrigo, W.W.P.; Athapaththu, A.M.M.H.
    Ribonucleic acid (RNA) is a polymeric molecule. It is implicated in coding and gene expression. Some medically important organisms such as viruses have only RNAs as their inherent material. To detect the viral diseases using molecular biological methods, it needs extraction of RNA from body fluids. There are several methods of RNA extraction, which require costly reagents and kits. Hence, the objective of this study was to optimize a low cost, in-house protocol for RNA extraction of viruses in clinical samples in order to facilitate disease diagnosis. Clinically confirmed blood samples, which were positive for Dengue Virus by NS1 antigen test, were taken for optimization of the two protocols. Two different RNA extraction protocols were used for the study to identify the most appropriate and reliable method with high efficiency. Trizol reagent, which was prepared in house was used in both protocols. Extracted RNA from both the protocols were quantified at 260 nm using a spectrophotometer. The RNA amount quantified from the spectrophotometer showed a result of 64 and 72 ng/ul from first and second protocols, respectively. In the first protocol, all the procedures were undertaken at room temperature (27-35 °C) but generally RNA is not stable at the room temperature. Therefore, RNA might have degraded due to lack of optimum conditions during the incubation, centrifugation and storage periods. In addition, if the RNA pellets were air dried completely, it becomes insoluble in RNase free water. Therefore, extracted RNA might not have been re-suspended completely in the solution. Those identified drawbacks were adjusted in the second protocol. Further, incubation temperature and time period (4 °C and 30 minutes) and centrifugation time (15 minutes), were modified to achieve stabilization, complete precipitation of RNA molecules and to prevent degradation by RNases. According to the above discussed facts, this study reveals that the second protocol is more suitable for RNA extraction ofviruses in clinical samples. Keywords: Ribonucleic Acid (RNA), Virus, Extraction