pcr-rflp (4)


The quality and quantity of the extracted DNA are two key aspects for a successful PCR (Polymerase Chain Reaction) amplification. Also, a reduction in time and cost required for DNA extraction are important. The aim of this study was to compare and optimise the performance of five different DNA extraction methods by boiling meat tissues from cattle, buffalo, sheep, goat, chicken, camel, horse and dog in PBS (Phosphate Buffer Saline), distilled water, alkaline lysis buffers 1, 2 or 3. The results indicated that the boiling of meat and its products in alkaline lysis buffers was the best method to extract crude DNA. The optimised crude DNA extraction protocol was coupled with PCR-RFLP (Restriction Fragment Length Polymorphism) analysis for meat species identification. The developed assay was tested on 53 commercial beef and mutton samples, out of which three samples were found to be adulterated.  

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Malayasian researchers have developed an assay to simultaneously determine 7 meat species (beef, buffalo, chicken, duck, sheep, goat and pork) in processed meat products. Species specific primers to the 7 species were designed, which target the mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (ND5) genes, to amplify short DNA fragments (73-263 bp) by PCR (polymerase chain reaction). These were then treated with 3 restriction enzymes ( FatI, BfaI, and HPY188I) to cut the amplicons down into smaller fragments, which were separated by gel electrophoresis. The bp (base pair) length and number of these fragments are unique to each of the species. The assay was tested against 25 non-target species to ensure specificity to the 7 target species, and the limit of detection was determined as 0.5% (w/w) in different matrices. The assay worked on heat treated meat products. A survey of local market meat products detected  buffalo DNA in 84% of commercial beef burgers and frankfurter products tested.

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Indian researchers have developed a protocol based on PCR-RFLP (restriction fragment length polymorphism) by treating a 515 bp fragment in the highly polymorphic mitochondrial D-loop region with a single enzyme Tsp5091 to give fragment fingerprints to identify 9 species of snapper. Individual species could be differentiated by 3–5 major bands. Very closely related species like Lfulvus and Lfulviflamma gave similar patterns due to high (94%) identity, while the other seven species were clearly differentiated. The method worked with frozen, cooked and fried fish, and was tested on samples of snapper purchased in a local market.              Read the abstract at: snapper identification                               

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Indian researchers have developed a PCR-RFLP method to authenticate 4 shrimp species of the family Penaeidae, namely, Litopenaeus vannamei, Penaeus monodon, P. semisulcatus and Fenneropenaeus indicus.  PCR amplification was performed targeting 16S rRNA/tRNAval region having an amplicon size of 530 bp using the specific primers for shrimps, 16S-Cru4/16S-Cru3. Subsequent restriction analysis with a single restriction enzyme, Tsp5091, yielded a distinct RFLP pattern for each species of shrimps having fragment sizes below 150 bp. The method works with processed shrimp products, and was validated with  commercial products.

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