A rapid test for detecting porcine DNA using loop-mediated isothermal amplification assay towards authentication of halal food
Increasing demands for high level of credibility in the food industry has triggered the attention of researchers to conduct research, aimed at finding an efficient and substainable solution to the global food fraud menace. Ethical and religious rights of Muslims are been infringed by this act. At pr...
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| Format: | Thesis Book |
| Language: | English |
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| Summary: | Increasing demands for high level of credibility in the food industry has triggered the attention of researchers to conduct research, aimed at finding an efficient and substainable solution to the global food fraud menace. Ethical and religious rights of Muslims are been infringed by this act. At present, porcine species specific polymerase chain reaction (PCR) is the method that is widely used in screening of food and pharmaceuticals. However, some limitations impede its smooth application. High financial burden is the most significant limiting factor of the PCR application amongst others. Therefore, this study focused on closing these gaps. The objectives of this study were to design porcine species-specific loop-mediated isothermal amplification (LAMP) primers based on mitochondrial DNA and to develop for the first time, a rapid test to detect porcine DNA by using LAMP assay. The method used was in accordance with standard LAMP assay development protocol. DNA samples from 10 different tissue types, obtained from eight animal species were used. Six sets of LAMP primers were manually designed based on porcine tRNA-Lys and ATPase Subunit 8 genes. Amplification was done within a water bath at constant temperature of 63'C. The results were visually detected after staining with SYBR green fluorescent DNA dye and illuminated on UV light. A total of 105 reactions performed with porcine DNA from several tissue types namely; skeletal muscle, fat, intestine, omentum, kidney, liver, cardiac muscle, lung, spleen and blood tissue were all amplified and detected visually. All results from this study were confirmed by running on 2% agarose gel electrophoresis revealing an expected LAMP amplicon band size of 237 bp. thus. Indicating 100% positive predictive value. An average time of 25 min was expended for both reaction and detection. Analytical sensitivity of the assay was remarkable high with a limit of detection of 0.03 fg. Spiked tissue samples containing as little as 1% (0.25 mg) of pork mixed with 99% (39.75 mg) cattle beef were amplified. The overall detection probability of the assay was 99.7%. Finally, a simply andaffordable assay for detecting porcine DNA towards halal food certification was developed |
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| Physical Description: | xiv, 82 leaves: ill. (some col.); 30 cm. |
| Bibliography: | Includes bibliographical references (leaves 59-70) |