Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction
A real-time polymerase chain reaction (PCR) assay with molecular beacon probe was developed for the detection of pork in raw states. The method combined the swine-specific primers and molecular beacon probe to specifically amplify a 119-bp fragment of porcine mitochondrial cytochrome b (mt-cyt b) ge...
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| Format: | Thesis |
| Language: | English |
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2012
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| Online Access: | http://psasir.upm.edu.my/id/eprint/39351/ http://psasir.upm.edu.my/id/eprint/39351/1/IPPH%202012%204R.pdf |
| _version_ | 1848849121850425344 |
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| author | Mohd Yusop, Mohd Hazim |
| author_facet | Mohd Yusop, Mohd Hazim |
| author_sort | Mohd Yusop, Mohd Hazim |
| building | UPM Institutional Repository |
| collection | Online Access |
| description | A real-time polymerase chain reaction (PCR) assay with molecular beacon probe was developed for the detection of pork in raw states. The method combined the swine-specific primers and molecular beacon probe to specifically amplify a 119-bp fragment of porcine mitochondrial cytochrome b (mt-cyt b) gene. Mitochondrial genes are present in multiple copies and thus ensure available targets even in
degraded samples. A pair of 18-nucleotide swine-cytb-specific primers were designed using Primer 3 Plus software. On the other hand, a 34-nt molecular beacon
probe was designed using Beacon Designer 4 software. The porcine specificity of the primers and probe were checked by basic local alignment search tools (BLAST) to avoid mismatches with other species. A specificity test with 10 ng DNA of nine common meat providing land and aquatic species yielded a Cq value of 18.70±0.12 to 19.08±0.06 only with the pork DNA in a 40 cycle PCR reaction, demonstrating
the swine specificity of the primers and probe. The swine-specificity was further confirmed in a binary mixture of pork and beef. The method detected 0.1% pork in binary pork-beef mixture with a Cq of 25.79±0.20. A sensitivity test with 10-fold serial dilution revealed that the assay can determine 0.0001 ng of porcine DNA with a PCR efficiency of 96% with a good reproducibility, precision and high correlation coefficient (r2=0.9989). The shorter length target (119-bp) and strong sensitivity and
specificity suggest the method can be used for the routine analysis of pork adulteration in raw meats. |
| first_indexed | 2025-11-15T09:45:22Z |
| format | Thesis |
| id | upm-39351 |
| institution | Universiti Putra Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T09:45:22Z |
| publishDate | 2012 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | upm-393512015-07-03T08:06:19Z http://psasir.upm.edu.my/id/eprint/39351/ Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction Mohd Yusop, Mohd Hazim A real-time polymerase chain reaction (PCR) assay with molecular beacon probe was developed for the detection of pork in raw states. The method combined the swine-specific primers and molecular beacon probe to specifically amplify a 119-bp fragment of porcine mitochondrial cytochrome b (mt-cyt b) gene. Mitochondrial genes are present in multiple copies and thus ensure available targets even in degraded samples. A pair of 18-nucleotide swine-cytb-specific primers were designed using Primer 3 Plus software. On the other hand, a 34-nt molecular beacon probe was designed using Beacon Designer 4 software. The porcine specificity of the primers and probe were checked by basic local alignment search tools (BLAST) to avoid mismatches with other species. A specificity test with 10 ng DNA of nine common meat providing land and aquatic species yielded a Cq value of 18.70±0.12 to 19.08±0.06 only with the pork DNA in a 40 cycle PCR reaction, demonstrating the swine specificity of the primers and probe. The swine-specificity was further confirmed in a binary mixture of pork and beef. The method detected 0.1% pork in binary pork-beef mixture with a Cq of 25.79±0.20. A sensitivity test with 10-fold serial dilution revealed that the assay can determine 0.0001 ng of porcine DNA with a PCR efficiency of 96% with a good reproducibility, precision and high correlation coefficient (r2=0.9989). The shorter length target (119-bp) and strong sensitivity and specificity suggest the method can be used for the routine analysis of pork adulteration in raw meats. 2012-04 Thesis NonPeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/39351/1/IPPH%202012%204R.pdf Mohd Yusop, Mohd Hazim (2012) Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction. Masters thesis, Universiti Putra Malaysia. Polymerase chain reaction Pork Bacon |
| spellingShingle | Polymerase chain reaction Pork Bacon Mohd Yusop, Mohd Hazim Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction |
| title | Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction |
| title_full | Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction |
| title_fullStr | Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction |
| title_full_unstemmed | Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction |
| title_short | Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction |
| title_sort | detection of raw pork targeting porcine-specific mitochondrial cytochrome b gene by molecular beacon probe and real-time polymerase chain reaction |
| topic | Polymerase chain reaction Pork Bacon |
| url | http://psasir.upm.edu.my/id/eprint/39351/ http://psasir.upm.edu.my/id/eprint/39351/1/IPPH%202012%204R.pdf |