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
Subjects:	Polymerase chain reaction Pork Bacon
Online Access:	http://psasir.upm.edu.my/id/eprint/39351/1/IPPH%202012%204R.pdf
Tags:	Add Tag No Tags, Be the first to tag this record!

id	my-upm-ir.39351
record_format	uketd_dc
spelling	my-upm-ir.393512015-07-03T08:06:19Z Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction 2012-04 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. Polymerase chain reaction Pork Bacon 2012-04 Thesis http://psasir.upm.edu.my/id/eprint/39351/ http://psasir.upm.edu.my/id/eprint/39351/1/IPPH%202012%204R.pdf application/pdf en public masters Universiti Putra Malaysia Polymerase chain reaction Pork Bacon
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	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
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
qualification_level	Master's degree
author	Mohd Yusop, Mohd Hazim
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
granting_institution	Universiti Putra Malaysia
publishDate	2012
url	http://psasir.upm.edu.my/id/eprint/39351/1/IPPH%202012%204R.pdf
_version_	1747811786890936320

Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe and real-time polymerase chain reaction

Similar Items