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M. Cahyadi, M.
Department of Animal Science, Faculty of Agriculture, Sebelas Maret University, Jl. Ir. Sutami 36A Surakarta 57126
Agriculture, Biological Sciences & Forestry Earth & Planetary Sciences

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Development of mitochondrial 12S rRNA gene for identification of dog and rat in beef using multiplex PCR Cahyadi, M.; Taufik, I. M.; Pramono, A.; Abdurrahman, Z. H.
Journal of the Indonesian Tropical Animal Agriculture Vol 44, No 1 (2019): March
Publisher : Diponegoro University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14710/jitaa.44.1.10-18

Abstract

The 12S rRNA gene is one of unique regions in mitochondrial genome usually used for phylogenetic studies and species identification. The objective of present study was to develop species specific primers from mitochondrial 12S rRNA gene for identification of dog and rat in beef by using multiplex PCR assay. Three primer pairs of mitochondrial 12S rRNA gene specific for bovine, dog and rat were designed and selected to evaluate their specificity and fidelity. Moreover, a total of twelve DNA samples extracted from meat tissue were also prepared to test those primers using simplex and multiplex PCR. The PCR products were then visualized using 2% of agarose gel under the UV light and three of them were sequenced. In addition, sequence data were analyzed using Clustal Omega software and BLAST. The result showed that simplex PCR assay successfully amplified DNA targets which are respectively indicated by 155 bp (bovine), 244 bp (dog), and 491 bp (rat) of DNA bands. Furthermore, DNA sample sequences were identically similar to reference sequence used in this study. Multiplex and simplex PCR analyses also indicated that these primer pairs specifically amplified DNA target for each species in the samples containing various species. The results suggested that designed primers in this study could be used to identify dog and rat in raw beef containing these species meat. Further experiment should be conducted using meat-processed products and commercial meat products as samples.
DETECTION OF PORK CONTAMINATION IN FRESH AND COOKED BEEF USING GENETIC MARKER MITOCHONDRIAL-DNA CYTOCHROME B BY DUPLEX-PCR Ni'mah, A.; Kartikasari, Y.; Pratama, A. D.; Kartikasari, L. R.; Hertanto, B. S.; Cahyadi, M.
Journal of the Indonesian Tropical Animal Agriculture Vol 41, No 1 (2016): March
Publisher : Diponegoro University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14710/jitaa.41.1.7-12

Abstract

By mixing with pork, beef adulteration is frequently found in the traditional  market that very disturbing Moeslem community in Indonesia. This study was conducted to detect pork contamination in fresh and cooked beef using genetic marker mitochondrial DNA cytochrome b (mt-DNA Cyt b) by duplex-PCR. A total of twelve samples was used in this study consisting six fresh meat samples and six cooked meat samples, respectively. Those beef and pork were bought from animal slaughterhouse and a supermarket in Surakarta. Cooked samples were prepared by boiling the meats in hot water at 100oC for 30 minutes. We designed pork contamination in beef in the level of 0, 1, 5, 10, 25%, respectively. The DNA genome was extracted and polymerase chain reaction (PCR) was performed using species specific primer to isolate mt-DNA Cyt b gene from the samples. The results showed that the DNA genome was successfully extracted from pork, beef, and contaminated meat samples. In addition, visualization of duplex-PCR on 1.5% agarose gel was able to detect pork contamination in both fresh and cooked beef up to very small proportion (1%). The existence of pork in beef was indicated with the presence of specific 398 bp DNA band. It can be concluded, duplex-PCR of mt-DNA Cyt b gene was very sensitive in detection of pork contamination in fresh and cooked beef.
Co-Authors A. D. Pratama, A. D. Abdurrahman, Zakaria Husein B. S. Hertanto, B. S. L. R. Kartikasari, L. R. Ni'mah, A. Pramono, A. Taufik, I. M. y. Kartikasari, y.