Browsing by Author "De Silva, D.P.D.C."

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    Identification of Horse Meat and Beef using a Polymerase Chain Reaction Based Method with Cytochrome b Gene
    (Uva Wellassa University of Sri Lanka, 2013) Munasinghe, M. M. E.; Bulumulla, P. B. A. I. K.; Fernando, T. S. R.; Samaraweera, A. M.; Nanayakkara, A. K.; De Silva, D.P.D.C.; Bandara, K.G.W.W.; Senaratne, S.G.
    Adulteration of meat and meat products are taking place in many parts of the world and it is believed that numerous such incidents have even occurred in Sri Lanka as well. Demanding meat and meat products are being adulterated with cheaper or unconventional meat types (eg. dog, horse or rat meat) with phenotypic similarities. This situation created a scenario where the food analyst from many developing countries needs to pay special attention to identify meat. A recent disclosure in UK about an adulteration of beef with equine meat created a paranoid situation, which drastically affected genuine brands produced by large companies and international food chains (Thomson, 2013). Hence, the adulteration must be prevented to ensure traceability of meat from farm to form. In general, molecular methods facilitate accurate and more reliable analysis of meat adulteration. Compared to nuclear DNA, application of mitochondrial DNA (mtDNA) in meat identification experiments provides many advantages, such as maternal inheritance and ubiquitous natures (Kvist, 2000). Thus, mtDNA can be used when the evidentiary material supply is limited. Among the Mitochondrial genes mitochondrial cytochrome b (Cyt b) gene has often used to detect the source of meat (Farias, et al., 2001). Hence, the aim of this study is to establish qualitative polymerase chain reaction method to detect horse meat adulteration in beef using mitochondrial DNA Cyt b region. Methodology Beef and horse (Equus ferus caballus) meat samples were collected from slaughter house at Dematagoda and from Faculty of Veterinary Medicine and Animal Science, at University of Peradeniya, respectively. Genomic DNA was extracted following the protocol as described in Abdel-Rahman et al. (2009) with modifications. First, 600 mg of tissue was homogenized in 6000 μL STE buffer and 48 μL of 10% SDS and 24 μL of proteinase K (10 mg/mL) was added. Then, the mixture was incubated at 37 °C overnight. After that, DNA was purified by equal and chloroform–isoamylalcohol (24:1), successively. Then, DNA was precipitated by adding chilled ethanol in the presence of 3 M sodium acetate. The resulting pellet was washed with 70% ethanol, air-dried and subsequently dissolved in 80 μL of TE buffer. Species specific primers were designed (horse forward (HF) - ATC ATC ACA GCC CTG GTA GTC GTA CAT, horse reverse (HR) - ATG TGG AGG GTG GGG ATG AGT GCT A, cattle forward (CF) - CAT CGG CAC AAA TTT AGT CG and cattle reverse (CR) - GAG CTA GAA TTA GTA AGA GGG CC) to amplify mitochondrial Cyt b gene of cattle and horse. The PCR products were electrophoresed on 2% agarose gel containing 0.5 µg/mL Ethidium bromide and were visualized and imaged using a UV trans-illuminator (Gel Dox XP+ system, BioRad) and gel documentation system (Image Lab 3.0, BioRad) to distinguish the species origin. Furthermore, to investigate the detection limit of the PCR system, DNA was extracted from 600 mg of beef which was mixed separately with 1%, 5% and 20% of horse meat. Results and Discussion MtDNA evolve at a much faster rate than nuclear DNA. At the same time different regions of the mitochondrial genome would evolve at different rates. Therefore, mtDNA maintain variable regions but with a certain level of conservation. Similarly, mitochondrial Cyt b gene contains both slow and rapid evolving regions with conservative and variable regions. The evolution of the Cyt b gene is prevented due to the functional restrictions in the conservative regions (Farias, et al., 2001). Therefore, Cyt b gene is used to identify horse meat from beef as the sequence variability in Cyt b gene between different species is extremely high.
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    Role of microorganisms against hydrocarbon contamination; Bioremediation
    (Uva Wellassa University of Sri Lanka, 2015) Arachchi, S.M.W.; Munasinghe, M.M.E.; Sabaragamukorale, S.T.; Abeygunaratne, S.S.; Rodrigo, W.W.P.; De Silva, D.P.D.C.; Dalpatadu, K.S.L.; Senaratne, S.G.; Gunathilaka, P.A.D.H.N.
    The development of human civilization has changed its path since the industrial revolution. Since then began the use of hydrocarbon sources as the primary energy source of the world. The use of oil as fuel has led to intensive economic development worldwide. Even though these compounds contribute to the global economy on massive scale they in turn have perilous effects on the biotic and abiotic components of the ecosystem. In the stages of oil refinement, transportation, storage and on daily activities, unavoidable oil spills take place in small amounts. However, the accidental large oil spills draw the attention of the public to find remediation solutions. The methods of remediation can be physical, chemical or biological or may be a combination of two or more of these techniques. Hydrocarbon utilizing bacteria, fungi and cyanobacteria have been found in soil, marine and fresh water ecosystems (Okoh, 2002). Although several countries have already used methods including microorganisms for bioremediation of petroleum spills, it has not been previously used in Sri Lanka. Therefore, the objective was to isolate indigenous bacterial strains from hydrocarbons contaminated soils to assess their potential for bioremediation and to develop a bio-product for bioremediation. Methodology Three sites with soil contaminated by different petroleum hydrocarbons were identified in Ceylon Petroleum Corporation, Sapugaskanda, Kelaniya, Sri Lanka. A total of 18 soil samples (6 from each site) were collected randomly by simple soil sampling method (American Society for Testing and Materials, 1998). A weight of 10 g of soil was diluted in 90 ml of 0.1% sterile Sodium pyrophosphate solution containing 30 g of sterile glass beads. After shaking the mixture for 1 hour at 175 rpm, the and were vortexed for 1 minute. A volume of 120 µl of each dilution was spread on Luria Broth (LB) agar medium and was incubated at 28 °C for 7 days. The colonies appeared were inoculated on a Bushnell Haas (BH) liquid and solid mediums supplemented with 50 µl of hydrocarbons followed by an incubation at 28 °C for 7 days. The identified colonies were subjected to genomic DNA extraction using the Phenol-Chloroform method. The extracted genomic DNA samples were sent over to Macrogen, Korea for 16S rRNA sequencing. The overnight grown bacterial cultures were centrifuged at 16000 g for 3 minutes at 4ºC. The pellet was resuspended in 200 µl of TE buffer and was vortexed and centrifuged at 16000 g for 1 minute at 4ºC and a volume of 1.5 µl of Protinase K was added and mixed. To this 20 µl (1/10) of 10% SDS was added, mixed well and incubated for 1 hour at 37 ºC. After the incubation, equal volume of Phenol: Chloroform (1:1) was added and centrifuged at 16000 g for 2 minutes at 4ºC. The aqueous layer was taken out without disturbing the protein layer and transferred into a fresh tube. A volume of 2V of 100% ice cold Ethanol and 0.1V Sodium acetate were added, mixed well and were incubated at 0 ºC for 1 hour. The solution mixture was centrifuged at 16000 g for 5 minutes at 4ºC. The supernatant was discarded and the pellet was dried and dissolved in 40 µl of nuclease-free water by tapping. For the selection of immobilizing agent, 10 g of autoclaved saw dust and rice husk each were mixed with 7.5 ml of Yeast Extract Glucose (YEG) broth separately and was autoclaved. Then the washed, pure bacterial cells were inoculated on to autoclaved rice husk and saw dust at room temperature and were incubated at 30 °C at 150 rpm for 5-6 days in a shaking incubator. The immobilized samples were washed with sterile saline water for 3 times and were inoculated on BH agar plates with diesel. Pure cultures of selected bacterial strains were inoculated with LB agar and were incubated over- night. A single colony of each bacterial strain was inoculated on 5 ml of LB broth. The cultured cells were centrifuged at 2000 g at 4°C for 10 minutes and the pellet was dissolved in 5 mL of phosphate buffer and re-centrifuged under the same conditions. Then the pellet was re-suspended in 5 ml of phosphate buffer. A mass of 14 g of autoclaved rice husk were mixed with 21 ml of YEG broth and was autoclaved. Then 2 ml of washed Bacterial cultures were inoculated on 2 g of autoclaved rice husk at room temperature separately and were incubated at 30 °C at 150 rpm for 5-6 days in a shaking incubator until a heavy culture develops. A volume of 20 ml of water was contaminated with 2 ml of diesel and 0.2 g of immobilized rice husk was added on top of the oil layers under sterile conditions. Turbidity and the time taken for the disruption of oil layer in the water were compared with a control.