Cover Image

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...

Full description

Saved in:
Bibliographic Details
Main Author: Mohd Yusop, Mohd Hazim
Format: Thesis
Language:English
Published: 2012
Online Access:http://psasir.upm.edu.my/id/eprint/39351/1/IPPH%202012%204R.pdf
http://psasir.upm.edu.my/id/eprint/39351/
Tags: Add Tag
No Tags, Be the first to tag this record!
id my.upm.eprints.39351
record_format eprints
spelling my.upm.eprints.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.
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
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.
format Thesis
author Mohd Yusop, Mohd Hazim
spellingShingle 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
author_facet Mohd Yusop, Mohd Hazim
author_sort Mohd Yusop, Mohd Hazim
title 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_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_sort detection of raw pork targeting porcine-specific mitochondrial cytochrome b gene by molecular beacon probe and real-time polymerase chain reaction
publishDate 2012
url http://psasir.upm.edu.my/id/eprint/39351/1/IPPH%202012%204R.pdf
http://psasir.upm.edu.my/id/eprint/39351/
_version_ 1643832398546206720
score 13.211869

Similar Items